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The Distribution, Pathogenicity and Population Dynamics of Pratylenchus thornei on wheat in South Australia.
Julie Margaret NICOL B.Ag.Sc. (Hons) University of Adelaide
Thesis submitted for the degree of Doctor of Philosophy
The Universtä Adelaide (Faculty of Agricultural and Natural" Resource Sciences) Department of Crop Protection.
January 1996 Contents
Preface
Title I Contents ü Summary vi Declaration ix Acknowledgements X Abbreviations xüi
Chapter I Introduction 1
Chapter 2 Literature Review 2.I Systematics and Distribution 3 2.2 Host Range and Distribution 3 2.3 Biology, Histopathology and Life Cycle 6 2.3.1 Biology 6 2.3.2 Histopathology 6 2.3.3 Life Cycle 10 2.4 Symptoms t2 2.5 Associations with Other Pathogens t3 2.6 Environmental Infl uences l5 2.6.1 Climate t6 2.6.2 Soil t7 2.6.3 Nutrition 18 2.7 Survival 19 2.8 Economic Importance 2t 2.9 Population Dynamics and Control Measures 25 2.9.I Chemical Control 30 2.9.2 Cultural Practices 32 2.9.3 Biological Control 34 2.9.4 Resistance and Tolerance 35
Chapter 3 General Laboratory Techniques 3.1 Nematorle Inocula 38 3.2 NematodeExtraction 39 3.2.1 Soil 39 3.2.2 Root 39 3.3 Nematode Counting 39 3.4 Staining Nematodes and./or Fungi 40 3.4.I Nematodes Only 40 3.4.2 Nematodes plus Fungi 40 3.5 Seed Sterilisation and Germination 40 3.6 StatisticalAnalysis 4I 3.6.r Statistical Designs 4I 3.6.2 Analysis of Variance and Error Bars 4I 3.6.3 Transformations 42 3.7 C,lassification of Soils used in Laboratory Experiments 42 lll
Chapter 4 støtewid.e sary.ey for P. thornei and p. neglectus ín the cereal Regions South Australia of 4.1 Introduction 43 4.2 Materials and Methods 44 4.3 Results 46 4.4 Discussion 48
Chapter 5 Multiplícatíon of P. thorneí and Development of a Resístance Assay Cereal and Non-Legumínous Hosts for 5.0 Generallntroduction 5l 5.1 Comparative Multiplication of p. thorneiover time 5.1.1 Introduction 52 5.1.2 Materials and Methods 52 5.1.3 Results 55 5.I.4 Discussion 58 5.2 Relation between p. thornei and p. neglectus 5.2.1 Introduction 6t 5.2.2 Materials and Methods 6T 5.2.3 Results 63 5.2.4 Discussion 65 5.3 Develop_ment of a Resistance Assay 5.3.1 Introduction 68 5.3.2 Materials and Methods 68 5.3.3 Results 70 5.3.4 Discussion 74 5.4 Modification of a Resistance Assay 5.4.1 Introd rction 76 5.4.2 Materials and Methods 76 5.4.3 Results 77 5.4.4 Discussion 79 General 5.5 Discussion 79
Chapter 6 Population Dynamics and Yield Relations ol P. thornei in the Laboratory 6.0 General Introduction 82 6.r Population parhogeniciry !4amic¡ and of p. thomei on Machete at25"C 6.1.1 Introduction 82 6.1.2 Materials and Methods 83 6.1.3 Results 84 6.1.4 Discussion 87 6.2 Soil Type-Relations p. p. with thornei and neglectus 89 6.2.I Introduction 89 6.2.2 Materials and Methods 89 6.2.3 Results 9I 6.2.4 Discussion 95 6.3 Population pathogenicitv Dynamics and of P. thornei on Machete at20"C 6.3.1 Introduction 97 6.3.2 Materials and Methods 97 6.3.3 Results 98 6.3.4 Discussion 103 IV
6.4 Population Dynamics and Pathogencity of P. thornei on V/arigal at2O"C 6.4.I Introduction 105 6.4.2 Materials and Methods 105 6.4.3 Results 106 6.4.4 Discussion Il4 6.5 General Discussion I 16
Chapter 7 Field Population Dynamics and Yíelil Relation ol P. thornei 7.1 Intoduction II7 7.2 Materials and Methods 119 7.2.I General Methods 119 7 .2.2 Sampling Device t20 7.2.3 Field Trial Layout t2r 7.2.4 Sampling Methodology for Initial Density t24 7.2.5 Plant and Nematode Cha¡acters Measured t26 7.2.5.I Plant Parameters Sampled 126 1.2.5.2 Nematode Variables Sampled 127 7.3 Results 7.3.I Plant Parameters Sampled t27 7.3.2 Nematode Variables Sampled 133 7.4 Discussion r37
Chapter I Plant Genetic Control and Possible Mechanisms of the P. thornei Resistance in Cereøls 8.0 General Introduction 148 8.1 Initial Penetration of Resistant and Susceptible Hosts 8.1.1 Introduction l4g 8.L.2 Materials and Methods 150 8.1.3 Results 151 8.1.4 Discussion I52 8.2 Inheritance of P. thorn¿i resistance in the wheat cultivar, AUS4930 8.2.I Introduction 153 8.2.2 Materials andMethods 154 8.2.3 Results 155 8.2.4 Discussion 159
Chøpter 9 NematodelFungal Interactions 9.1 Introduction t63 9.2 Materials and Methods r64 9.3 Results r66 9.4 Discussion t79
Chøpter I0 Final Discussion and Conclusions 185 v
Appendices
Appendix A Experimental Data for the Statewide Distribution Survey of P. thornei and P. neglectus in the cereal growing regions. 193
Appendix B Experimental Data for the Field Population Dynamics and Yield Relations of P. thorn¿i on cereals for the 2year trial at Tanunda. 201
Appendix C Preliminary Investigation into the Molecular Distinction of P. thornei and P. neglectus. 2O4 C 1.0 General Introduction 2O5 C 1.1 Extraction of DNA from nematodes C 1. 1. 1 Introduction 206 Cl.l.z Materials and Methods 2O6 C1.1.3 Results and Conclusions 209 Cl.2 RFLP Hybridisation Analysis C1.2.1 Introduction 209 cl.2.2 Materials and Methods 2rr cl.2.3 Results 2t2 cl. 2.3 General Conclusion 2t3
Appendix D Morphometrics of South Australian populations of P. thornei and P. neglectus males and females. 2I5 D1.0 Abstract 216 D 1. I Introduction 217 DI.z Materials and Methods 217 D1.3 Descriptions 218 Dl.4 Discussion 2Ig
References 224 vl
The root lesion nematode (Pratylenchus thornei) has been identified as a damaging
pathogen on cereals worldwide and within Australia in Queensland and New South Wales. In South Australia, P. thornei and P. neglectus have been found, but their
importance to the cereal industry has yet to be defined. Although the research reported
here focused primarily on P. thornei, several experiments involved P. neglectøs. The
major objectives of the project were to determine the distribution of both Pratylenchus
species in South Australia, to study the field and laboratory population dynamics of P.
thornei in relation to wheat yields, to determine its host range on a variety of cereal and
non-leguminous hosts and to identify possible sources of nematode resistant wheat
cultivars/varieties. The involvement of root rotting fungi with the nematode in wheat
disease was studied in preliminary experiments.
The statewide survey for P. thornei and P. neglectus in soil and plants from the cereal
growing regions in South Australia showed that there was a gOVo chance of finding one
or both species of nematode in a given soil type. P. neglect¡1.ç was more commonly
found in sandy soils, while P. thornei tended to be associated with clay soils, although
this distinction was not definitive. The survey confirmed that both nematodes had a wide
host range.
An assay for screening cereal and non-leguminous hosts was developed. Plants could be
effectively screened over two months instead of five, using plants grown in a sandy soil
in small polyethylene tubes inoculated with a non damaging initial density of 400 P.
thornei. From the plants examined, varying degrees of nematode multiplication were
evident for both nematode species. The majority of commonly cultivated Australian wheats were highly susceptible to P. thornei. Triticale, rye, oats and durum were moderately susceptible to resistant, while the non-leguminous hosts showed suggested resistance to P. thornei. Similar results were obtained for P. neglectus. However, in some instances differences in nematode multiplication between some varieties/cultivars within the T. aestivum species were evident. The variety AUS4930 was one of the least
susceptible wheats tested for P. thornei, but the most susceptible for P. neglectus.
Laboratory studies on yield relations and population dynamics on wheat found that P.
thornei significantly affected many growth variables. In general, low initial densities at
early stages of growth (up to 5 weeks) were associated with a stimulus of many plant
growth variables, possibly a host response to damage. However, higher initial densities
significantly reduced many growth variables, verifying that P. thontei damages wheat in
its own right.
The field population dynamics and yield relations of P. thornei were examined in a two
year trial established in the Barossa Valley in South Australia. P. thornei caused
significant yield losses up to 38Vo on commonly cultivated South Australian wheats,
however the initial P. thornei density associated with yield reductions was seasonally
variable. Two suspected resistant wheat varieties, AUS4930 and GS50A, were
confirmed as resistant in the field, and a common South Australian wheat (Warigal) was
found to be highly susceptible. The population dynamics of P. thornei followed the
general pattern of nematode behaviour, with low initial densities associated with high
multiplication and higher densities with reduced multiplication. However, the
equilibrium density for P. thornei was approximately 10,000 P. thonteil2O0g OD soil,
whioh was well above previously documented P. thornei thresholds on cereal crops.
Preliminary studies investigating the mechanism of P. thorn¿i resistance showed that in
both wheat varieties AUS4930 and GS50A the resistance acted post-penetration. Genetic
inheritance studies with AUS4930 and a commonly grown South Australian wheat
suggested further selection of both parents was necessary to define accurately the genetic
basis of the resistance.
There were synergistic associations of wheat damage with P. thornei and P. neglectus and two commonly occurring South Australian root rotting fungi, Fusarium acuminatum vut and Microdochium bolleyi. It will be necessary to further investigate such associations, particularly before adoption of resistant cultivars, because fungal infection might lower resistance.
From this study P. thornei is considered to be economically important in South Australia.
The polyphagous host range and polycyclic nature of the nematode will make effective control of the nematode difficult, but not impossible. The two wheat varieties, field selections of GS50A from Queensland and 4US4930, originally from Iraq, offer potential sources of resistance to P. thornei in the field. The influence of root rotting fungi in combination with Pratylenchøs on resistance needs to be carefully considered for successful nematode control, as well as the inherent differences in cultivar reaction to the two nematode species. IX
Declaration
This work contains no material which has been accepted for the award of any other degree or diploma in any univeristy or other tertìary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.
I give coqle-ry t9 tltis copy ofmy thesis, when deposited in the University Library being available for loan and photocopying.
Signed Date X
Acknowledgements
This PhD has been finally finished and could not have been done so thoroughly without
the help of those named below. The meury people listed have all been worth their weight
in gold, and I have been fortunate to have encountered them. They all have been an
integral part of forming the "Prat. Task Force".
I would firstly like to acknowledge my supervisors, Nematologist, Associate Professor
John Fisher for his advice, guidance and support during the practical component of the
project. Dr Kerrie Davies and Trevor Hancock have been invaluable during the last year
of the project, Trevor and Lynne Giles for their help on the statistical analysis of the
multitudes of data and Kerrie for her constant support, advice, constructive criticism and
nematological input. I also thank the current Head of the Crop Protection Department,
Professor Otto Schmidt, former retired Head of Plant Pathology Department, Professor
Harry Wallace and a scientist far removed from Plant Pathology, Geologist Dr Colin
Winsor for their constructive reading of my thesis manuscript.
I gratefully acknowledge financial support from the Grains Research Development
Corporation who enabled this study to occur. I wish to thank several people within the
Corporation, in particular Dr Rob Brown, Tom Cootes, John Petschack and Cathy
Stewart for their constant interest and assistance.
There are many other people which I would like to mention who have been invaluable in
allowing the project to address the objectives:
The soil survey was successfully achieved with the help of many participants, the District
Agronomists of Department of Primary Industries in South Australia (Trevor Dillon,
Tony Craddock, John Hannay, David Lewis, Brenton Growden, Peter Fulwood,
Margaret Evans (Minnipa Research Centre)), private consultant Nick Hillier and numerous farmers involved in Agricultural Bureaux and Land Care Groups, in particular 'Williams, Dean Don Greig, Bruce Heddle, Kevin Shopp, Roger Scholz and Bill
Blumson of Eyre Peninsula. XT
The laboratory screening and field work involved acquiring seed from sources far and
wide. I gratefully acknowledge all the people who were able to assist me in this pursuit;
Franky Green (SARDI), Sue Pelham and Dr Andrew Ban (Department of Primary
Industries), Dr Cath Cooper (University of Adelaide), Dr Frank Ellison (University of
Sydney), Trent Potter (Department Primary Industries), Dr John Thompson and Dr Paul
Brennan (Queensland Wheat Research Institute) and Michael Mackay (Australian'Winters
Cereal Collection).
Special thanks to the two farmers involved with this study, Mr Peter Grocke of Tanunda for allowing to use his paddocks over several years for this study. His time, interest, assistance and allowing me to multiply the nematode on his land has enabled an intense field study to be conducted. I also thank Mr Peter March for allowing me to also use his paddock at Mallala, although the mighty gods above were displeased with this choice, with a drought and mice plague successfully destroying any possibitity of obtaining a 'Waite crop for the two consecutive years of trial work. I would also like to thank the
Institute Wheat Breeding Unit, in particular Dr Tony Rathjen for the numerous conversations (lively debate mostly) and support, also the assistance of Michael K¡oehn in the planting and harvesting of the trials conducted. I also thank Mark Barber (formerly
SARDI) for assistance with spraying the trial and Sharyn Taylor for sharing her vast field trial experience with root lesion nematode.
Thanks also to several people who were integral parts of the molecular and morphological taxonomic studies. In particular, Janine Lloyd (University of Adelaide) for drawing nematode specimens, Professor Otto Schmidt and Dr Ulrich Theopold, Sassan Asgari ,
Natalie Dillon (University of Adelaide) and Dr Dara Whisson (SARDI) for advice and constructive criticism of the molecular diagnostics.
I would like to acknowledge those people who worked collaboratively with certain aspects of the project, in particular Dr Paul Brennan (Queensland V/heat Research xlr
Institute), Michael Kroehn (University of Adelaide), Abdolhossein Taheri (University of
Adelaide), Sharyn Taylor (SARDÐ and Dr Vivien Vanstone (University of Adelaide) for
their time and effort to combine resources and grey matter in the pursuit of knowledge.
To the small but cohesive nematological network at the Waite, known as the "Toad
Team" steered by Kerrie Davies, thank you all for your interest, support and for also
going partially blind studying microscopic organisms and most importantly having the
firm belief that Nematologists count! (a lot, too much?). Furthermore I am indebted to
several people within the Department for their invaluable assistance and ongoing patience
and friendship, in particular Kerrie Davies, Heather Fraser, Abdol Taheri and Otto
Schmidt.
I must mention the "typing pool", namely friends who were once close until this task was
bestowed upon them, Kate Byron-Scott, Fiona Withers, Heidi Wilkinson, Suzanne
Forster, Grace Nicol and Ben Rose for all their help in the final stages of thesis
preparation. In addition to these godsends I would like to thank Sean Rowe, Nick
Hillier, Tom Hawker, Phillipa Sharpe, Darren Graetz, Veronica Hall, Natalie Dillon,
Cate Hill, Ian Delaere and Wanja Kinuthia for their constant presence and eternal
friendship, all of who made this task that much more attainable. I must also thank my
wonderful Aunties, the seven Roberston girls, who all in their wee \ryays helped. Finally
I gratefully acknowledge the never ending love and support of my parents, Margaret and
Barry, and of my brother Douglas and sister-in-law Grace. I also thank my little niece
Jessie for removing me from the realms of scientific academia, aiding the disorganisation of my study and keeping me sane. xlll
Abbreviations
ANOVA analysis of variance cm centimeúe CRD completely randomised design d.f. degrees of freedom DDW double distilled water DNA deoxyribose nucleic acid f.wt. fresh weight o Þ $am û Þ gravitational force ha hectare kg kilo gram L line M molarity (g/L) m mefre
m.s. mean squ¿ìre mg/rnl milligrams per millilitre ml millilite mM milli molar mm millimetre nm nano metres NSW New South Wales OD oven dry PDA potato dextrose agat pers. comm. personal communication Prob. F probability psi pounds per square inch RCBD randomised complete block design SARDI South Australian Research and Development Institute SDV/ sterile distilled water SED standard error of difference SPD split plot design tJha tonnes per hectare USA United States of America V volts v.r. va¡iance ratio vs' versus pto xlv w/w wet weight t degree Celsius p micron ps micro-gram pl micro-litres pm micro-metre prnl micro-millilitre i'ì'il çþQ|lcr l ",l ',,,.ir,îriiil,
Chapter L Introduction
The world population is growing at an unsustainable rate with our resources being
pushed to their limits. The population is expected to increase from 6.1 billion by the
turn of the century to 8.2 billion by 2050 with more thangOVo of this increase expected
from developing countries. Worldwide, rice, wheat, corn and potatoes are the staple
foods with wheat being the primary food in 43 countries (Reitz, 1967). An increase in
wheat productivity would help to ameliorate the impact of this population increase.
In Australia, there are more than 45,000 farmers annually producing about 15 million
tonnes of wheat of which over 85Vo is exported, making it Australia's third largest
export earner. On a global scale, Australia is the fourth largest wheat exporter,
contributing about llVo of the world market (National Farmers Federation, 1995).
South Australia contributes about l4vo of the total Australian production.
The Australian production per hectare of wheat is less than that of competitors, and despite an active research and extension program the production per hectare has not risen significantly in the last 20 years (National Farmers Federation, 1995). As a consequence current research is attempting to identify and rectify this problem. A contributor to the problem may be the root lesion nematode (Pratylenchus sp.) which reduces wheat yields in both southern and northern wheat belts of Australia. There are over 63 species in this genus (Loof, I99l), most of which are destructive pests on many plants. The nematodes move through the soil from root to root (Dropkin, 1989), producing characteristic narrow, elongated lesions on root surfaces or in the cortex.
In Australia, six species of Pratylenchus have been recorded on wheat but only two species, P. thornei and P. neglectus, are considered of economic importance principally 2 Chapter I InÛoduction due to their widespread distribution (J. Thompson; A. Pattison; V. Vanstone and S.
Taylor, pers. comm.). Both nematodes are morphologically similar, polyphagous and polycylic. P. thornei is known to markedly limit yield of wheat worldwide and within
Australia is a known problem in the northern and eastern wheat belts and is also of importance in Southern Australia. P. neglectus is considered a potential problem only in South Australia. Terms such as "long fallow disorder" and "wheat sickness" have been used to describe the heavy yield losses caused by P. thornei on the Darling Downs in Queensland (Thompson ¿f al., 1981) and NSW (Doyle et aI., 1987). Preliminary evidence from Victoria and South Australia suggests P. thornei limits yield of wheat
(Eastwood et al.,1994; Nicol, 1991).
This study was undertaken to contribute to our understanding of the role of P. thornei in cereal production in South Australia. The main objectives of this research were to;
' determine the distribution of P. thornei and P. neglectus within the southern cereal belt.
' evaluate the host efficiency of the commonly cultivated cereal and non-leguminous crops to P. thornei.
' investigate in the field the relationship between nematode population density and yield
in several cereal species.
' identify and investigate the possible mechanism and mode of inheritance of resistance in wheat to P. thornei.
' conduct preliminary investigations into the possible role of some root-rotting fungi in
their association with P. thornei on the production of wheat.
In addition to the above, the first Australian investigation of the morphometrics of the two species P. thornei and P. neglectus was made and preliminary attempts were made to distinguish both species using molecular techniques. 3 Chapter 2 Literature Review
Chapter 2 Literature Review
2.1 Systematics and Description
The genus Pratylenchas (Tylenchida: Pratylenchidae), established by Filipjev in 1936,
which parasitizes a wide range of plant hosts has been difficult to classify taxonomically,
with between 45-63 species depending on which key is used (Loof, 1991). On wheat, at
least six species have been recorded: P. thornei (Fortuner, 1977), P. neglectus
(Anderson & Townshend, 1976), P. zeae (colbran & McCulloch, 1965), P. crenatus
(Loof, 1978), P. mediterraneus (Corbett,1970)and P. pinguicaudatus (Corbett, 1970).
In Australia, only three of these have been recorded on cereals: P. thornei, P. neglectus
and P. zeae (Colbran & McCulloch, 1965).
Pratylenclzzs species are commonly known as root lesion nematodes. They are migratory obligate endoparasites with an average length of 0.5mm, (range 0.3 to 0.8mm)
(Southey, 1978). Pratylenchøs species have a long slender vemiform shaped body with a short, strong stylet (Southey, 1978).
In South Australia, two species, P. thornei and P. neglectu,r, are commonly associated with cereal growing areas (Ch.4). The morphometric similarity of Pratylenchus species makes taxonomic identification a major impediment to sound ecological studies (Stirling
& Stanton, 1993). Work described in Appendix D shows that the Australian forms of P. thornei and P. neglectus are indistinguishable from those in Europe, Africa, North
America and the United Kingdom.
2.2 Host Range and Distribution
A host plant provides sustenance to a nematode parasite and allows reproduction
(Caveness, 1974). P. thornei is polyphagous and attacks roots of plants from at least 18 botanical families ( Table 2.1 ), including the basic food crops of cereals, legumes and 4 Chapter 2 Literature Review potatoes. Fortuner (1977) cites wheat (Triticum aestivum L.) as a primary host of P
thornei.
FAMILY HOSTS WITHIN FAMILY Chenopodiaceae Beta vulgaris, Spinacia oleracea Composrtae Chrysanthemum sp., Helianthus spp., Lactuca sativa, Cucurbitaceae CitruIlus lanatus Cupressaceae Chama ecyparis spp., Cupressus spp. Crucifêrae Brassica oleracea vatbotrytis, Iberis sp., Raphanus sativus .Encaceae Arc to stap hy lo s pun g ens Fagaceae Quercus spp. Gramrneae Ec hino c h lo a frument ac e a, H o rde um v ul g ar e, P anic um sp., S ac c hør um fficinarum, Sorghum bicolor, Triticum aestivum, T.durum, Tritico secale, ka mays Grossula¡iaceae Ribes rubrum Juglandaceae JuRIans regia Leguminosae Cicer arietinum, Crotalaria juncea, Glycine max, Lens culinaris, Lupinus sp., Medicago sativa, Medicago rigidula, Phaseolus vulgaris, Pisum s ativ um, Trifo Ii um r e p en s, Vic i a fab a, V i c i a s qtiv a, V i gna un g uic ulata,Vi gna s ine sis, Lrlraceae AIIium cepa Linaceae Linum usitatissimum Pinaceae Pinus spp. Rosaceae Þ'ragaria sp., Malus pumila, Prunus spp, Prunus domestica, Prunus persica var. nectarina, Pyrus communis, Rosa spp.
Solanaceae Ly c op e rs ic on e s c u I entum, S o lanum tub e ro s um 'l'heaceae Camellia sinensis Umbelliferae I)aucus carota, Coriandrum sativum Vitaceae Vitis vinifera
Table 2.1 : Host Range of P. thornei ananged by families [Derived from Forruner (1977), Greco et.al.(1984; 1988), Loof (1978) and O'Brien(1982), Evans & Webb (1989), Thompson (private comm. 1993)l
P. thornei has a world wide distribution but records are few. It has been reported in :
Europe : England, Denmark, France, Spain, Greece (Thompson, private coÍtm. 1993);
Italy, Yugoslavia, Holland, Belgium and Germany (Fortuner, 1977). Africa : canary
Islands, South Africa, Egypt (Thompson, private comm. 1993); Morocco (Ammati, l98l); Algeria (Troccoli et al., 1992). North America : Senora in Mexico, California in USA (Loof, 1978). South America : Venezuela (Loof, 1978). Asia : India
(Fortuner, 1977); Korea (Thompson, private comm. 1993); Japan (Loofl 1978); and 5 Chapter 2 Literature Review
Pakistan (Maqbool, 1987). Middle East : Syria (Greco et al., 1988); Iran, Cyprus
(Thompson, private comm. 1993): Israel (Loof, 1978) and Australia : (Baxter and
Blake, 1967; Fortuner, 1977).
P. thornei has been recorded on wheat in the following countries : Europe : Yugoslavia
(Fortuner, 1977); Italy and Mediterranean region (Lamberti, 1981). Africa : Morocco 'West (Ammati, 1987), Algeria (Troccoli et aI., 1992). North America : Senora in
Mexico and Utah in USA (Fortuner, 1977) Asia : India and Pakistan (Maqbool, 1987)
Middle East : Northern Negev in Israel (Orion et al., 1982) and Syria (Greco et a1.,1988) and Australia (Fortuner, 1977) .
Within the Australian wheat belt, P. thornei is known to occur in four States and is predominantly found in the heavy-textured soils. At present, the distribution encompasses Dubbo in the south of NSW, north to the Darling Downs and to Chinchilla in the north of the Queensland cereal belt (Thompson, private comm. 1993). In
Victoria, P. thornei occurs in clay and loam soils at Nhill in the'Wimmera and at Elmore and Rochester in the North-Central area (Thompson, private comm. 1993) and Charlton and Horsham (J. Fisher, pers. comm.). In South Australia, P. thornei was identified by
Fisher in 1956 (J. Fisher, pers. comm.). It has been recorded in the Adelaide metropolitan area (Grandison, L972; Singh, 1984) and is widespread throughout the cropping regions of South Australia (S. Taylor, pers.comm.; V. vanstone, pers. comm.).
The closely related species P. neglectur occurs in temperate regions in Europe, Canada,
United States, Japan, South Africa and North-Western India (Townshend & Anderson,
1976). P. neglectus has a widespread distribution in the cereal areas of Victoria
(Meagher, 1970), Vy'estern Australia (J. Stanton, pers. comm.), the North-West wheat belt of Queensland (Mcculloch & Thompson, unpub. data), NSrw (R. Mcleod, pers. comm.) and South Australia (Stynes & veitch, 1983, s. Taylor, pers. comm.). Reports 6 Chaptàr 2 Literature Review
of. P. neglectus in Queensland are sporadic compared with P. thornei (Thompson, private comm. 1993). In the cropping regions of South Australia, P. neglectus ís widespread on the Eyre Peninsula and is also commonly found in combination with P. thornei on Yorke Peninsula, in the South-East and to a lesser extent the Lower to Upper
North (Ch. 4).
2.3 Biology, Histopathology and Life Cycle 2.3.1 Biology
P. thornei is a primary root parasite of wheat (Fortuner, 1977). There is some difference in opinion relating to the form of parasitism; Dropkin (1989) considered
Pratylenchøs spp. to be endoparasitic while Fulton et al. (1960) thought they were both ectoparasitic and endoparasitic. Reproduction in the genus Prarylenchas is bisexual
(Dropkin, 1989), but males of P. thornei are rare (Fortuner, 1977). Females of P. thornei do not have functional spermathecae (Suatmadji; in Thompson, private comm.
1993) and reproduction is by mitotic parthenogenesis. A closely related species P. mediteraneus (formerly known as P. thorner) has similar morphology, however the presence of males has led to its recognition as a new species (Orion et al., 1984)
2.3.2 Histopathology
Pratylenchøs spp. invade the root cortex and kill cells during feeding, resulting in brownish elongated lesions (Dropkin, 1989). Hence they are known as root-lesion nematodes. P. thornei invades roots in a non-random fashion, being attracted to parts of the root already invaded and their subsequent reproduction results in groups of nematodes at intervals along both seminal and nodal roots (Baxter and Blake,1967). As a consequence, nematodes are concentrated in localised areas and cause damage to restricted parts of the root. The percentage of P. thornei invading wheat roots at a particular site decreases as the nematode numbers increase due to a shortage of locations '7 Chapter 2 Literature Review for penetration and/or interference between individual nematodes (Baxter & Blake, te67).
Studies by Baxter and Blake (1968) on invasion of wheat roots by P. thornei under field and aseptic conditions established that P. thornei alone is pathogenic. In both aseptic and field conditions, P. thornei causes lysis of the parenchyma cells resulting in cell wall disintegration and formation of cavities in the cortex after three weeks. Following destruction of the cortex, the epidermis is sloughed off exposing the uninvaded stele which is often necrotic. Lesions (0.5-2mm) in the proximal parts of the seminal roots were noted after six weeks, but did not affect the structure of the stele. In stained root segments, P. thornei was usually located in the cortex lying parallel to the long axis of the root. Orion and Lapid (1993) found similar histopathology with P. mediteraneus, which is closely related to P. thorn¿i. Males, females and larvae of P. mediteraneus invaded the cortical cells resulting in root collapse. Scanning electron microscope studies of P. mediteranues onVicia sativa revealed that the nematodes invaded the roots at the root hair region by forming a clear "drilled" hole in the root epidermis and the cortical parenchyma, suggesting enzymatic lysis combined with mechanical force (Kurppa and
Vrain, 1985). A dense layer of bacteria surrounded the invaded region. Inside the cortex, the nematode moved within the parenchymal tissue destroying the cell cytoplasm.
Movement of the nematode through cell walls again suggested use of enzymatic activity.
The surface of the invaded region appeared as a lesion on which the root hairs were shed and the epidermis v¿as densely punctured. At the edges of the lesion, abnormally long root hairs were observed. In cross and longitudinal sections, aggregation of nematodes was observed in the root cortex where the plant tissue was completely destroyed. Eggs were deposited in cavities apparently formed by the nematode. Orion and Lapid (1993) described P. mediteraneus as a migratory endoparasite although there is evidence of ectoparasitic activity causing damage to root apices. It is possible that P. thornei may have similar behaviour. 8 Chapter 2 Literature Review
Probably no two species of Pratylenchus produce an identical plant response to parasitism. Even plant reactions which appear outwardly similar may differ biochemically. A wide range of histopathological reactions may arise from the enoÍnous number of possible combinations of plant species or cultivars and nematodes and the varied physiology of both (Southey, 1986). Cortical invasion and necrosis is also associated with other species of Pratylenchus. Ogiga and Estey (1975) found that P. penetrans invaded the cortex and caused necrosis on turnip and corn and refer to other authors with similar findings on alfalfa, pea, peach, apple, carrot, celery and strawberry.
P. coffeae on Musa sp. also has similar histopathology (Pinochet, 1978). However, the histology of P. thorn¿i differs from that of the closely related species P. neglectus, which is also a pathogen of wheat. Studies by Anderson and Townshend (I976) found that P. neglectus invades in a non-random manner, being attracted to the root tips in particular. This is probably a chemokinetic response. P. neglectus does not feed predominantly on the cortical cells, but rather externally on the meristematic tissue behind the root cap, causing root elongation to cease, after which the nematode invades and migrates to more mature areas of the root (Anderson and Townshend, 1976). Studies on wheat by Kimpinski ¿r al. (1976) found that P. neglectus invades the seminal roots first, then the crown roots and observed that there were much lower numbers in the latter.
P. fallax, also pathogenic to wheat, is another species closely related to P . thornei, and it has a non-random invasion similar to that of P. neglecrzs (Corbett, 1972). P. fallax invades the main roots of wheat, barley and sugarbeet at the root tips, the region of root hair development, and at points where the laterals emerge. Unlike P. thornei, browning of the endodermis produced visible lesions before the cortex became necrotic. However, like P. thornei, P. fallax did not penetrate the stele.
Studies by Townshend et aI. (1989) on the histopathology of P. penetrar?,r on alfalfa roots, indicate the nematode moves through the cortex intracellularly, with nematodes once again lying parallel to the stele. The cortical parenchyma cells are penetrated and fed 9 Chapter 2 Literature Review upon, containing only cytoplasmic debris after 48 hours of feeding. The proximal cells had increased tannin deposits, degenerate mitochondria, increased numbers of ribosomes and no internal membrane structure. Often the endodermis had collapsed and contained massive tannin deposits on the inner cell wall and cell lumen.
Similar studies by Vovlas and Troccoli (1990) of P. penetrans in broad-bean roots found the nematode located entirely inside the cortex and generally longitudinal to the vascular cylinder with stelar tissues unaffected. Nematodes fed intracellularly causing extensive rupturing of cell walls, cavities and thickening of cell walls, or necrosis of the cells around feeding sites. There was no associated hyperplasia or hypertropy of the cortical cells but thickening of two to three layers of cells adjacent to the nematode were observed.
Some species of Pratylenchus appear to cause different histopathology in different hosts.
P. penetrans invaded the endodermis and stele in corn, but the endodermis was a barrier to invasion in apple, peach, celery, strawberry and pea (Ogiga and Estey, 1975). Root exudates have been considered the most likely stimulus attracting Pratylenchøs to a host plant (Baxter and Blake, 1967; Wallace,19'14; Vy'allace, 1989). Production of toxins, enzymes and unidentified compounds by both the host and nematode may play a role in root invasion and destruction by respective Pratylenchus spp. P. penetranr was found to induce lateral root formation in turnip and corn (Chen et a1.,I963). It has been suggested that this response is associated with a phenolic defence mechanism of the host, which inactivates the hydrolytic enzymes of the nematodes and signals growth response by regulating indole acetic acid (ogiga and Estey, 1975). In peach, strawberry and celery, root necrosis was found to extend beyond the infection court of P. penetrans
(Baxter and Blake, 1968) suggesting toxins produced directly or indirectly by the nematode induced necrosis in advance of the feeding site. However, there are few histochemical investigations of plants infected with P. thornei. Such studies are essential for the understanding of the disease complex between pathogen and host. l0 Chapter 2 Literature Review
2.3.3 Life Cycle
Invasion, movement through and exit from roots, mây be accomplished by both adult and juvenile forms (Fulton et aL, 1960). Root-lesion nematodes possess four similar developmental stages, each separated by a moult, with juveniles resembling the adult forms except for maturation of the reproductive system (Fulton et aL, 1960). The first juvenile stage and first moult occur in the egg, and the emerging second-stage juveniles move about in the soil or enter roots (Dropkin, 1989). Subsequent juvenile stages develop until the fourth moult from which the juveniles emerge as adults. Females require a host plant to oviposit (Larson,1953), but juveniles of P. thornei can moult into females independent of a host (Thompson, private comm. 1993).
Figure 2.1 illustrates the general disease cycle for Pratylenchøs spp., which can be split into 3 components (Fulton et aI.,1960):
1. Penetration in which the larvae and adults of various stages enter and leave the roots of susceptible hosts.
2. Dissemination of eggs, larvae and adults. The female, with or without fertilisation, may lay eggs singly or in small groups inside infected roots (Fortuner,
1977); the eggs hatch or are released into the soil following root breakdown (Agrios,
1988).
3. Oversummering in which the egg stage is probably most persistent, but most other growth stages are quiescent in the soil or when hosts are unavailable (Fulton et al.,
1960).
P. thornei attacks crops in the winter growing season and subsequently survives over drought conditions in a desiccated state (see section 2.7) untlI reactivated by rainfall the following winter (Grandison and'Wallace, L974; Glazer and Orion, 1983). Pratylenchus spp. are thought to leave the root for the surrounding soil environment when the roots become hard and inhospitable (Fortuner, 1977). ll Chapter 2 Literature Review
The life cycle of Pratylenchø.s spp. is completed 45-65 days (Agrios, 1988), depending on the species, temperature, as well as other factors contributing to this variability
(Fulton et aL,1960). Optimum conditions for development are species-specific. Few studies on the life cycle and optimum conditions of Pratylenchus spp. have been made.
Larson (1959) found the complete life cycle of P. thornei took 40-45 days at 27"C. This included two days for the female to penetrate the root, one day to lay eggs, seven days for the egg to hatch into a second stage juvenile, seven days for the next moult to a third stage juvenile, four days to the third moult, fourteen days until the fourth moult was complete and about eight days until the female coÍrmenced laying. However, Siyanand et aI. (1982) found the life cycle (egg to egg) of P. thornei required 25-29 days under laboratory conditions at 3È.2'C. Baxter and Blake (1968) observed appearance of second stage juvenlles 24 days after wheat had been inoculated with adults, and the work of Orion et al. (1979) indicates the optimal soil temperature for reproduction of P. mediteraneus is between l8"C and22"C.
l.tmlod! hrcdl4 \ a@l R@l sygtcm ol corllr hcollhy plqnl 3h rool d¡rcclly \ lnvodôd ond odull! li!!uos r@ls lurn brown
rgsr. 3rd trpll
plonl l Ncmolodes trd m¡grolo lh.
\Nemlodos þow 'dccoying roolr
',/ sl / IIst rool! my br ) ond lh.¡r Eoos o?c lo¡d or kt hd lMdsd @llcor rÉl;o$d ¡n loil I Slogr lorvo l- ¿'
Fig. 2.1 : Life cycle of the root lesion nematode, Pratylenchas spp.(source: Agrios, 1988)
Larson (1953) demonstrated the reproduction and completion of the life cycle of P. thornei in wheat roots. Thompson (private comm. 1993) suggests that P. thornei t2 Chapter 2 Literature Review completes several generations within the life span of a single wheat crop and infers that nematode multiplication follows the compound interest epidemiological model described by Van der Plank (1975). Such exponential multiplication in the early stages of growth has been recorded by Baxter and Blake (1968). Thompson (private coûrm. 1993) further suggests that in a monoculture system P. thornei survives about six months fallow between successive wheat crops. This would mean that the population increases in a series of intemrpted compound interest curves until population equilibrium is reached.
2.4 Symptoms
As mentioned in Section2.3, root lesions are the main symptom for hosts infected with
Pratylenchus spp. Thompson (private comm. 1993) found wheat plants severely damaged by P. thornei with dark brown lesions within the cortex of the roots and sometimes with discoloration extending to the stele. At lower population levels or with tolerant wheat cultivars lesions were not seen readily, but roots had general light-brown discoloration compared with white uninvaded roots (Thompson, private comm. 1993).
Portions of plants affected by Pratylenchus spp. above ground do not express definitive symptoms. In general, damaged plants appear stunted and unthrifty, unspecific symptoms which could be caused by restricted or non-functional root systems, drought or mineral imbalance in the soil (Fulton et al., 1960). Effects of P. thornei on growth, yield and vigour have not been demonstrated consistently. Van Gundy et al.(1974) noted infected wheat plants were rarely killed but usually stunted and chlorotic, sometimes with necrosis at leaf tips accompanied by reduced tillering, size and number of ears, and only one head being produced instead of two to four per plant. Ear sterility was a symptom in
Yugoslavia, while in India plants appeared sickly with poor growth (Fortuner, 1977); however, infestation occurred without browning or lesions. Severe stunting and shrunken grain are reported for wheat in the USA infested with P. thornei (Thorne,
196I), but Larson (1953) was not able to demonstrate any adverse effects of P. thonnei in inoculated soil or in infested fields in California. 13 Chapter 2 Literature Review
In Australia, Thompson's studies (private comm. 1993) in Queensland seem in part agreement with the work of Van Gundy et al. (1974). Wheat attacked by P. thornei appears stunted with few tillers and yellowed lower leaves with green and upright upper leaves. Affected wheat wilts prematurely compared with barley or nematode-free wheat under slight moisture stress. Doyle et al. (1987) record similar findings of stunting, restricted tillering and yellow lower leaves in NSW, particularly in old farming country where wheat has been grown regularly for many years while adjacent fields were often unaffected.
2.5 Associations with Other Pathogens
Pratylenchus spp. are successful in penetrating the root beyond its protective barriers and as a result infected roots are almost always invaded by secondary microorganisms including pathogens (Mountain,1954). Subsequently, rotting and further deterioration of root tissues occurs such that a disease complex between the nematode and secondary pathogen is established which may play a significant role in reducing crop yields (Powell, I91l).
The most widespread disease assoication with Pratylenchus spp. is with fungi, particularly rootrot complexes (Powell, I97l). Fungi found associated with the roots of plants containing PraryIench¿¿s spp. include Verticillium, Phytophthora, Pythium,
Perenospora, Aphanomyces, Cylindrocarpon, Fusarium and Trichodermø (Dropkin,
1989). Thorne (1961) found that oats and maize, infested with P. thornei were more susceptible to severe attack by smut (Ustilaginales). Synergistic interactions occurred between Pratylenchrs sp. andVerticllium dahliae andlor Erwinia carotovora (Faulkner and Skotland, 1965; Krikun and Orion, 1977; Riedel and Rowe, 1985; Santo and Huan,
1992). Benedict and Mountain (1956) found Pratylenchus spp. associated with the fungus Sclerospora, causing root rot of sugar cane. 14 Chapter 2 Literature Review
With respect to wheat, the work of Van Gundy et al. (1974) in Mexico suggests other soil organisms may contribute to the damage caused by P. thornei. They found
Rhizoctonia solani, Pythium sp., Penicillium spp., Alternaria sp. and Bipolaris sp. in the roots of declining plants infected with P. thornei. They proposed that the field disorder was caused by a disease complex of P. thornei and R. solani. Such a complex involving R. solqni and P. neglectus has been shown to cause almost twice the yield reductions on wheat than with the nematode alone (Benedict and Mountain, 1956). In
South Australia, P. neglectus has been associated with R. solani causing bare patch on wheat and barley (de Beer, 1965). Siddiqi (1986) also found a negative association of P. brachyurus and R. solani on peanuts, together causing greater damage than either organism alone. The work of Taheri et al. (1994) found P. neglectus increased severity of lesioning on wheat roots with the following fungi: Pyrenocheta terrestris, Pythium irregulare, Fusarium oxysporum,and Gaeumannomyces graminis in combination with
Fusarium equiseti. These are all found commonly in the cropping regions of South
Australia.
In addition to interactions with fungi, some bacterial infections are enhanced by
Pratylenchus spp. Dropkin (1989) found Agrobacterium catsed more damage to roses in the presence of Pratylenchus than on its own. The disease complex of P. penetrans and Pseudomonas spp. caused significantly more growth reduction on alfalfa than the sum of either pathogen alone, suggesting a synergistic relationship (Bookbinder et al.,
1982). The plant height, root weight and root score of apple was significantly reduced when P. penetrar?s was combined with Bacillus subtilis (Utkhede et al., 1992).
Similarly bacterial wilt in tomato caused by Pseudomonas was enhanced by Pratylenchus
(Dropkin, 1989). 15 Chapter 2 Literature Review
Complexes between nematodes occur, although few are cited in the literature. P. zeae, which is a known pathogen of cereals, has a synergistic association with the nematode
Tylenchorhynchus vulgaris on maize (Upadhyay and Swarup, 1981). Recently, much work has investigated the association of migratory and sedentary nematodes and suggests that the effects are varied and dependent upon species of nematode. For example,
Umesh and Ferris (1994) found P. neglectu.r suppressed M. chin'voodiby reducing egg production, final population levels, reproductive index and in addition reduced the damage caused to potato and barley. Field observations of Lasserre et al. (1994) indicated that a reduction in population densities of P. neglectus on wheat coincided with the development of the cereal cyst nematode (Heterodera avenae) in wheat roots. In support, Pratylenchu.r spp. and H. avenae were also negatively correlated on barley
(Esmenjaud et al., 1990). Other associations include M. incognira, which is known to suppress P. brachyurø,r on soybean (Herman et al., 1988), and P. penetrans on tomato
(Estores et aI., 1972).
2.6 Environmental Influences
The impact of a nematode community on a population of host plants is the result of all the interrelationships both within and outside the community, with the total environment, including interactions with the host plants. For example, the physico-chemical aspects of the environment constitute one set of parameters governing nematode populations
(Norton, 1979).
Despite Fortuner's (.1977) view that P. thornei has a "cosmopolitan" distribution, certain ecological conditions favour its activity, reproduction and survival. The rate of population increase and the final equilibrium population density of P. thornei wlll depend on the environment as well as such factors as the initial nematode density and the host species. In discussing some of the ecological factors influencing P. thornei , the l6 Chapter 2 Literature Review following subsections on Climate, Soil and Nutrition will concentrate on the nematode with wheat as its host.
2.6.1 Climate
As previously noted in Section 2.3, P. thornei is active in the winter (growing season) in
South Australia and subsequently survives drought conditions (over summer) in a desiccated state until reactivated by rainfall in the following autumn winter (Grandison and Wallace,1974). P. thornei appears to be associated with the semi-arid zones of
Utah (Thorne, 1961), Mexico (Van Gundy et aI., 1974) and southern Australia (Baxter and Blake, 1968; Grandison,l9T2), which all experience a Mediterranean climate characterised by winter rainfall and summer drought, with hot summers and cool and mild winters (Sale, 1982). However, Grandison's (1972) studies implied that temperature, rainfall and precipitation-evaporation ratio were unimportant in influencing the population and density of P. thorn¿i. Similarly, Thompson et al. (1989) found that high populations of P. thornei caused yield loss independent of seasonal conditions in
Australia.
In contrast, work with the closely related species, P. mediteraneus found low moisture levels was a major ecological factor affecting the nematode multiplication in Northern
Negev, where Orion et al. (1984) recorded the highest populations during a drought year and the lowest numbers in exceptionally wet years. Further, irrigated soil was a sub- optimal environment for the nematode which had no effect on yield under these conditions (Orion et aI., 1984). However, opposing results have been found with P. neglectus and P. penetrans. Low soil moisture was associated with fewer P. neglectus
(Kimpinski, 1972), and under soil moisture conditions of 115 bars, reproduction, root invasion and survival of P. penetrans were greatly suppressed (Kable and Mai, 1968). 11 Chapter 2 Literature Review
The inference of this suggests further work on the effect of climatic components need to be examined for P. thornei.
The work by Glazer and Orion (1983) showed that P. mediteraneur was able to withstand desiccation for up to 7-8 months, remaining infective. Moistening the soil activates dormant stages of P. mediteraneus or causes hatching of eggs in soil or plant remnants (Orion et a1.,1979). Under experimental conditions, wheat plants kept at 14'C during the early part of growth were not significantly affected by P. thornei ,but plants kept at 25"C showed maximum reduction in yield (Van Gundy et al., l9l4). Similar studies with P. neglectus on wheat found maximum penetration and movement at 20"C
(Umesh and Ferris, 1992), while the life cycle was completed most rapidly at25-30"C.
However, Townshend and Anderson (1976) found that greatest invasion and penetration of P. neglectus in maize roots was at 30"C. Orion et al.(1979) recorded the optimal temperature for P. thornei reproduction was 18 - 22"C. This suggests that differing physiological activities of the nematode have different optimal temperatures which are determined according to the host and surrounding environment.
2.6.2 Soil
Both soil type and depth influence P. thornei. Fortuner (1977) stated that P. thornei preferred heavier textured soils. Most global records of P. thornei are associated with clay soil: in Utah on heavy clay loams (Thorne, 1961), Holland on heavy soils (Loof,
1960), Mexico on fine textured clay soils (Van Gundy et aI., 1974), Netherlands,
England and Wales on clay soils (Loof, 1960; Corbett, 1970).
In Australia, P. thorn¿i is also associated with heavy soils. Thompson (private comm.
1993) found P. thornei in heavy cracking clay soils or vertisols from Dubbo in NSW to
Chinchilla in Queensland. In the northern cereal belt on the Darling Downs in l8 Chapter 2 Literature Review
Queensland P. thornei occurred on a full range of heavy textured soils including self- mulching, massively-structured black earths, grey clays and red loam soils (Thompson,
1989). Doyle et aL (1987) noted P. thornei on dark heavy-medium textured clays in northern NSW. In South Australia the distribution of P. thornei is not clearly defined.
Grandison and Wallace (1974) concluded that soil type was one of the main factors governing the distribution and abundance of. P. thorn¿i in Adelaide. Their studies found the hot dry summers markedly inhibited reproduction of P. thornei in sandy soil, but in clay soils higher moisture retention reduced such inhibition. The cool wet winters increased nematode reproduction in both sand and clay soils, but clay soils may be subject to waterlogging. Grandison (1972) concluded higher numbers of P. thornei were associated with clay than with sand soils in the Adelaide Metropolitan a¡ea.
P. thornei has been found from the topsoil to depths of 1.2 metres (Doyle et aL, 1987), with maximum numbers generally located between 30-60cm. In Queensland wheat fields, Thompson (private comm. 1993) and Doyle et aI. (1987) noted P. thornei was found at depths of 20-60cm particularly after a period of fallow. This is in contrast to the soil distribution of ËL avenae which occurs largely in the top 30cm of soil, and as a result control of P. thornei is more difficult. Depth distribution of the nematode may be influenced by the host. In Egypt, P. thornei occurred at 40cm depth under fallow, but moved upwards in the soil profile when sugarcane was planted (Fortuner, l9l1).
2.6.3 Nutrition
Nitrogen, commonly applied as ammonia or urea, has important effects on plant growth and P. thornei, however there is confusion in the literature on what the possible effects are. Van Gundy et al. (1974) claimed that nitrogen application offered some measure of r9 Chapter 2 Literature Review control, but only when the P. thornei population was near the threshold for economic damage (42 P. thornei /100cc soil). If the population was 5-7 times higher, nitrogen had no effect. Kimpinski (1912) found that the concentration of ammonium nitrate was correlated with fewer numbers and lower densities of P. neglectus in wheat roots. In addition, the effects on the disease complex of R. solani and P. neglectus was lessened by the addition of nitrogen fertilizer (deBeer, 1965). However, Thompson (private comm. 1993) found the rate of response in wheat yield to increased nitrogen fertilizer was less in soil infested with P. thornei than in soil treated with aldicarb (nematicide).
Thompson (1987) demonstrated that wheat roots infested with P. thornei take up soil nitrogen less efficiently than uninfested roots. However, growth of such infested roots was stimulated by fertilizer application, leaving higher populations of the nematode to attack subsequent crops. Doyle et al. (1987) found wheat yields in northern NSW were not significantly increased by the addition of nitrogen, and Orion et al. (1984) found no change in numbers of. P. mediteraneus with the use of 150kg/tra nitrogen.
Potassium and phosphorus fertilizers did not significantly increase wheat yields in the P. thornei -infested sites of northern NSW (Doyle et aI., 1987). Similarly, number and density of P. neglectus weÍe not altered by the addition of phosphorus and potassium to wheat (Kimpinskí, 1972), and populations of P. penetrans on red clover and alfalfa were not affected by potassium fertilization (Willis,l976). Again wheat yields were not significantly increased by application of copper, magnesium, boron, manganese, molybdenum or sulphur in a P. thornei infested site in NSW (Doyle et al., 1987), but zinc did increase yield.
2.7 Survival
The closely related species, P. mediteraneaus is only active in winter in non-irrigated wheat fields in Israel (Orion et aI.,1979), emphasising the need for an efficient survival 20 Chapter 2 Literature Review mechanism for the nematode, especially during the summer periods before subsequent reactivation by rainfall in the following winter. AII stages of P. thorn¿i survive desiccation (Thompson et al., 1981), during which the nematode is thought to enter a state of "anhydrobiosis". P. mediteraneaus have been observed to coil about themselves, shrink the body wall, and distort somatic muscles (Glazer and Orion, 1983).
Adaptations of all organisms to anhydrobiotic survival are manifested by a reduction in metabolic rate ('Womersley, 1987).
Thorne (1961) suggested that P. thornei migrated into the soil when roots became inhospitable and remained there until the next crop was planted. In an experimental area kept fallow for 5 years,SVo of the initial P. thornei population survived (Thompson and
Clewett, 1990). Survival of P. thornei in 2009 soil samples was reducedby SOVoby drying ftom 19.5Vo to 5Vo moisture and/or high temperatures (Baxter and Blake, 1968).
At temperatures of 40"C, mortality was induced within a two week period. Similarly, only 5Vo of the original population of P. thorn¿i survived in an aerated steam treated soil for 30 minutes at 50'C but was completely eliminated at 7O'C (Thompson, private coÍrm. 1993).
Heavy clay based soils, commonly found associated with P. thornei, may give more protection against heat than lighter textured soils, presumably because of moisture retention, hence may be associated with nematode survival (Thompson, private comm.
1993). In Queensland vertisols where stubble is burnt and retained, the greatest number of P. thornei occurred at a depth of 30cm (Haak et al., 1993). Studies on P. mediteraneøs in northern Negev of Israel, with a similar climate to Australia, showed little annual variation in population density (Orion et al., 1979). As suggested for P. thornei, P. mediteraneus is known to survive the unfavourable hot dry summer by entering an anhydrobiotic state (Glazer and Orion, 1983). Anhydrobiosis is induced 2l Chapter 2 Literature Review
when the relative humidity is reduced to 97.lVo. Only 3Vo of nematodes survived three
cycles of desiccation and rehydration. This has implications for fallowing in winter,
where the nematode population could be reduced by 80Vo because of intermittent wetting
of soil (Orion et al., 1984). S. Taylor (pers. comm.) found that several false breaks
appeared to reduce P. neglectus populations in the field in South Australia. Neutral lipids
in P. mediteraneus are accumulated by juvenile stages in unfavourable conditions (Storey
et a1.,1982) and may have a role in survival
When Baxter and Blake (1968) extracted P. thornei from soil, nematodes were inactive
and irregularly shaped at first but slowly became active. Thompson (1989) reported P.
thornei collected from dry fields can survive in undiminished numbers for at least two
years when stored at ambient temperatures. Viability of P. thornei decreased rapidly
during the first five weeks of storage and more slowly during the next fifty weeks, with
the loss being increased at higher temperature and moisture (Baxter and Blake, 1968). P.
neglectus also appears to survive desiccation (Townshend and Anderson, 1976).
Survival was best at2"C with mortality increasing with increasing temperature and soil
moisture. Survival of adults and 4th stage juveniles was greater than 3rd and 2nd stage juveniles (Townshend and Anderson, I976). P. neglectus can be considered "freezing
susceptible" and cannot survive sub-zero temperatures. It declined slowly over 15
months at low moisture to less than half the initial population (Meagher, 1970).
2.8 Economic Importance
As previously discussed in section2.3, P. thornei invades both seminal and nodal roots
and causes cortical degradation. Cortical loss in wheat is thought to reduce the
absorptive capacity of roots of certain grasses (Jacques and Schwass, 1956 ; in Baxter
and Blake, 1968). Simmonds and Sallans (1933) showed loss of seminal roots in wheat can reduce grain yield, but loss of nodal roots is more significant. However,
Krassousky (1926) indicated that on a weight-for-weight basis, seminal roots absorb 22 Chapter 2 Literature Review
twice as much as nodal roots. Thompson (private comm. 1993) found that P. thornei
severely limited the uptake of available nitrogen from the soil. Baxter and Blake (1968)
suggested that P. thornei would reduce yields in crops grown during the dry season, if a
high population was present in the soil at seeding. P. mediteraneus redtced capability of
root systems to absorb water and nutrients from the soil, subsequently causing water
stress and thereby affecting yield (Orion et aL.,1984).
Severe wheat decline in northern NSW and the Darling Downs in Queensland is associated with P. thornei (Doyle et aL, 1987; Colbran and McCulloch, 1965;
Thompson, private comm. 1993). Thompson and Clewett (1986) reported that P.
thornei caused yield losses up to 50Vo in wheat cultivars on the Darling Downs.
Thompson (private comm. 1993) noted that with intolerant wheat cultivars such as
Gatcher and Banks the yield loss was higher with up to 85Vo reduction. Although well
fertilized crops are less affected by P. thornei (section 2.6.3), high yielding varieties can
still cost an average loss of 0.5 tonnes/trectare (Thompson et aI., 1981). Populations of
P. thornei in Queensland exceeding 5OO/200g soil in any of the upper layers of the profile
constitute the economic threshold for intolerant wheats (Thompson, 1993). In northern
NS'W, Doyle et aI.(1987) observed reduction in wheat yields of 50Vo in paddocks
cropped with wheat alone for more than ten years. In South Australia, aseptic P. thornei
on wheat reduced dry weight of plants by 50% after 9 weeks in the laboratory (Nicol,
I99I), strongly suggesting P. thornei limited yield. Preliminary field work by Taylor
and McKay (1993) indicate densities of 1.5 P. thorneilg soil before sowing caused yield losses up to 70Vo in wheat. The work of Eastwood et al. (1994) in Victoria showed P.
thornei has the potential to reduce wheat yields by at least 44Vo and smaller amounts in other crops. It was estimated that per nematode per g soil P. thornei caused approximately 2Vo yield loss in the wheat cultivar Meering. Care should be taken with the P. thontei damage estimates recorded on wheat because most of these documented 23 Chapter 2 Literature Review yield losses were obtained using nematicides which may have influenced the growth of the plant and the microflora of the soil environment.
In countries other than Australia, P. thornei has been associated with major yield reductions of wheat in Utah, USA (Thorne, 1961) and Senora, Mexico with an economic threshold of 42 P. thorneillÙÙcc soil (Van Gundy et al., 1974). P. mediteraneus is also associated with severe yield reductions of wheat in the arid areas of Negev in Israel
(Orion et aI., 1984). P. neglectøs damages winter wheat in France when densities approach 3000/9 root (Lassene et aI., 1994). Laboratory tests have indicated that P. neglectus is damaging on cereals (Griffin, 1992; Mojtahedi et al., 1992; Umesh and
Ferris, 1994). In South Australia, Taylor and McKay (1993) found nematode densities up to 4 P. neglectøslg soil but these did not result in yield loss. However, A. Taheri
(pers. comm.), found at high initial densities P. neglectus caused up to 20Vo yield reduction of the wheat cultivar Machete.
Although P. thornei is associated with major yield loss, there are also other economic aspects to be considered. As previously documented in section 2.2, the host range of P. thornei is wide and includes an extensive range of important plant species associated with wheat rotations. Several legumes, especially chickpea (Cicer arietinum ), soybean (Glycine max ) and various other grams and cowpea (genus Vigna ) favour nematode multiplication. Among the cereals, maize (Zea mays ) and Triticale (Tritico secale) in particular favour nematode multiplication, whereas barley (Hordeum vulgare ) and sorghum (Sorghum sp.) are associated with few nematodes (Thompson, private comm.
1993). The significance of this is a loss in flexibility of crop choice to the producer, such that some coÍrmon practices of legume/wheat rotations in certain areas of the grain belt may no longer be a viable cropping sequence. However, crops such as sorghum may be substituted for legumes in wetter areas. In the more marginal areas of South Australia, 24 Chapter 2 Literature
opportunities for crops other than wheat are few thereby reducing the opportunities for
control through crop rotation.
Some farmers have attempted to control P. thornei by applying high rates of fertilizer,
but results in increased growth of weeds (Thompson and Clewett, 1988). Thompson
(private comm. 1993) noted that the need to control weeds by greater herbicide usage
increased the cost of production, or if not controlled led to a greater weed seed bank for
subsequent crops. This has resulted in a change from winter to summer crops on some
properties in Queensland.
The northern cereal belt supplies 25Vo of total wheat production in Australia, and the
presence of P. thorn¿i has been a major factor in farmers seeking to use alternative crops
(Thompson, private comm. 1993). So significant has the impact of P. thorn¿i been in
Eastern Australia that control mçasures are actively being researched. Similar research
has been conducted in Mexico by Van Gundy et al. (1974) who described a pest
management approach involving the use of variety selection, nitrogen fertilizer, planting
in cool soil (15'C) and crop rotation (avoiding wheat after wheat) as the most practical
solution to control P. thornei on a coÍtmercial scale.
The closely related species P. mediteraneus was controlled successfully on wheat in
Israel; with yield increases of 4O-9OVo attained by biannual fallowing and 5O-7OVo by the
use of soil fumigation (Orion et aL, 1984). Other Pratylenchzs species associated with
cereals, P. pinguicaudatus, P. fallax and P. crenatus in England (Corbett, 1970) and P. zeae in Queensland (Colbran and McCulloch, 1965), have not been investigated with respect to yield-reducing potential. 25 Chapter 2 Literature Review
As discussed previously, Prarylenchus species are kno\¡/n to be associated with other soil organisms, particularly fungi. The fungus R. solani has often been associated with high numbers of P. neglectus in crops which yielded poorly in South Australia (Stynes and Veitch, 1983). The preliminary work of A. Taheri (pers. comm.) suggests that fungi could be a major determinant of the degree of damage caused by P. neglectus in South
Australian cropping regions. A similar situation may also exist for P. thornei.
The following section provides some insight into possible control measures of P. thornei. However, the type of control used is ultimately determined by the farmers' financial and technical resources (Dropkin, 1989). At present there is no current adequate control of P. thornei and research is only preliminary.
2.9 Population Dynamics and Control Measures
The impact of plant parasitic nematodes on plant health and crop yield varies with biogeographic location, cropping sequence and intensity, cultivar selection, soil characteristics and nematode community structure (McKenny and Ferris, 1983).
Although Pratylenchzs is capable of multiplying for several generations during a single season, they spread only from plant to plant due to their relative immobility. It is unrealistic to attempt to design methods to eliminate plant parasitic nematodes.
Practically, control is adequate if the population remaining after harvest is smaller than would cause harm if the crop \üere planted again (Jones and Kempton, 1978).
Therefore, control of a particular nematode requires sufficient understanding of the nematode population dynamics, biology, ecology and interactions with other organisms.
Models of nematode population and plant yield are mathematical statements and computer programs that represent portions of presumed reality (Dropkin, 1989). The aim is to 26 Chapter 2 Literature Review predict nematode population and crop yields based on limited information about a particular situation. If the initial density of a nematode is known when a crop is planted then prediction of the final density at the end of the growing season can be made, and again at the start of the following year (Dropkin, 1989).
In order to study population dynamics nematologists need to obtain adequate estimates of numbers of nematode present. Sampling plans must be custom made for many different
situations since the relationship between the number of sample cores and relative error changes in response to many factors, including nematode species, field size, crop and
soil type (McSorley and Parrado,1982). Along with the sampling accuracy, the specific proportion of nematodes extracted using a particular technique and soil type is different
and needs to be established.
Frg.2.2 is a standard population curve for nematodes. The curves are uncomplicated by
emigration, immigration or the persistence of individuals not participating in reproduction. The scales of P¡ and P¡ are the same so a line drawn at 45" through the
origin is where neither population increase or decrease occurs, namely the multiplication rate is xl. As P¡increases the multiplication rate increases to its maximum and then
decreases to x1 or less, largely because of increasing competition between individuals
and decreasing food supply (Jones and Kempton, 1978).
Many authors (Barker and Olthof, 1976; Ferris, 1981; Jones and Kempton, 1978;
Oostenbrink,1966; Seinhorst, 1965) have investigated damage functions in annual crops by relating a parameter of plant performance to a single estimate of nematode population
density and age structure. A generalised model for damage is a linear regression of plant
growth against log-transformed initial nematode population density. Seinhorst (1965, 27 Chapter 2 Literature Review
1973) produced models based on theoretical consideration then tested them in a series of careful experiments. The model, based on the general biology of nematodes involved data collection from a wide range of samples including low to high nematode densities.
His model is based on the competition curve of Nicholson and assumed that "the average nematode" does not vary as population density changes and the ability of nematode to cause damage at high densities is unaltered (Dropkin, 1989).
The model of Seinhorst is illustrated in Fig.2.3. The yield of a crop under attack by a nematode is the sum of the low yield when nematodes are at the maximum level known for that crop plus an increment that depends on the particular nematode population observed. This increment (1-m) where m is the minimum yield, is affected by the proportion of roots that escape infection. This proportionZ is raised to an exponent. The exponent consists of the differences between the observed density of nematodes and that at which no damage results (Pi-T), where P¡ is the initial population density and T is the tolerance limit. From such models the economic injury level and economic threshold can be determined. The Economic Injury Level is the density of a particular nematode species that will cause a yield loss equal to the cost of nematode control, while the
Economic Threshold Level is the density used to determine the probability of economic injury and the need for nematode control. It may be equal to, or less than, the economic injury level (O'Brien and Stirling, 1991).
Other approaches in modelling plant parasitic nematodes have involved exploring the
"ceiling" reached by nematode populations on hosts (Dropkin, 1989). Nobling and
Ferris (1986) assumed each habitat to hold a "carrying capacity" for a particular nematode, implying that the environment can support a certain size population and when this is reached the population remained more or less constant. Intraspecific and interspecific competition for a limited resource is a major density dependent factor 28 Chapter 2 Literature Review constraining population growth (Duncan and Ferris, 1982,1983). In annual crops such as wheat, with relatively short growth cycles, logistic type depressions in multiplication rates are observed in the influence of P¡ (initial population) on the P/Pi (final population/initial population) ratio (Seinhorst, l97O; Jones and Kempton, 1978; Ferris ,
1985). Such models of seasonal multiplication are used for nematode management decisions. The objectives of such models is to predict final population size even though the dynamic interactions between nematode and host plant have been essentially ignored
(Brown and Kerry,1987). Measurements taken over a range of initial population densities indicate a density-dependent relationship between seasonal multiplication rate and initial population density. The maximum multiplication rate is seen at low initial densities when resources are unlimited. As initial population increases, the multiplication rate decreases owing to increasing competition between individuals, and a decreasing supply of food. At higher population densities the equilibrium level may be reached, at which the final and initial nematode populations are equal. Brown and Kerry (1987) reported that the work of Evans and Fisher (1970), Seinhorst (1970), Johnson et al.,
(1974), Jones and Kempton (1978) and Nobling and Ferris (1986) fit this general model.
1 logi:l rc crtrt \
I Eq
I 2 + o¡exuol À o ¿o
o 2 l 4 5 Loq(Pir t) Fig. 2.2 : The relationship between preplanting (P¡) and post-harvest population densities (Pr) of asexual and sexual species. The upper, logistic curve is the bne that would hold if the food luppry remained constant. El, log^iltic equilibrium point; Eq, observed equilibrium point. (Source: Jones and Kempton, 1978). 29 Chapter 2 Literature Review
Crop compensates for r any damage caused too Tolerance level of crop
of nematode numbers ô above which yield loss becomes _J 75 significant UJ ; Yield decreases as nematode l-m numbers increase l-rJ 50 F Yield loss reaches a maxlmum J 25 t-t_d m
T I N I TIAL NEMATODE DENSITY
Fig. 2.3: The relationship between relative plant growth and number of nematodes; Y=m * (l-m)ZPi-T . For Pi>T, y=l.0 for PicT, where Y=relative yield, m=mimimum yield, T=tolerance limit, Z is a constant reflecting nematode damage, Pi is the initial population density. (After Seinhorst, 1965. Source: Brown and Kerry, 1987 and O'Brien and Stirling, I99l).
The above models are only useful if the information from which they are derived is accurate. This information is essential for the implementation of management practices for nematode control, whether they be chemical, cultural, biological control or the use of resistance and tolerance or a combination of approaches, commonly termed integrated control. However, the method of control will be ultimately determined by the farmer's financial and technical resources (Dropkin, 1989). Two criteria can be used to judge whether control measures are successful. First, the yield increase by plant growth and the second, the population density of the pest after harvest (Jones and Kempton, 1978).
In the following sections some possible control measures of P. thornei will be considered. 30 Chapter 2 Literature Review
2.9.1 Chemical Control
Nematicides not only protect the crop to which they are applied but may leave few nematodes in the soil for the next crop, resulting in residual yield increases of up to 0.5 tonnes/hectare in P. thornei infested sites (Thompson ¿t al. ,I98I). There are several problems associated with the use of nematicides. P. thornei, as previously mentioned, can complete several life cycles in the span of a single wheat crop so that nematicides may not be fully effective (Doyle et aI., 1987). Also P. thornei appears to be associated with heavier soil and to depths of 1.2m (Doyle et aL, 1987), the amount of nematicides required will be too high and expensive to be viable in most cases. Furthermore, the use of nematicides is becoming increasingly undesirable because of environmental concerns they are associated with.
Nematicides can be categorised into three major groups; fumigants, organophosphates and carbamates. Considering fumigants, Ethylene dibromide (2.7-IO.8L/ha) was found to have no effect in reducing P. thornei numbers or increasing yield (Doyle et a1.,1987).
However, Methyl bromide increased wheat yields ftom 32-78Vo (Thompson, private comm. 1993; Doyle et al., 1987). Van Gundy et al. (1974) reported a side effect of bromine toxicity induction in Mexican wheat. Work with the closely related species, P. mediteraneausby Orion et aI. (1984) found soil treated with Metham sodium resulted in
5O-70Vo yield increase and 90Vo redtction of nematode populations, but its use was only viable in inigated areas. Thompson (private comm. 1993) found that broad spectrum fumigants controlled P. thornei, but they proved toxic to mycorrhizal fungi and may kill much of the microbial biomass which play an important role in nitrogen supply. The fumigants Chloropicrin (22Ùkglha) and Methyl-isothiocyanate liberator Dozomet
(450kg/ha) were less effective than aldicarb (l0kg/tra) (Thompson, private comm. 1993).
The more specific nematicidal fumigant D-D (1, 3-Dichloropropene) at l87L/h4 led to yield increases on wheat plants in P. thorn¿i infested soil in Mexico (Van Gundy et al.,
1974), and Telone 11 (1, 3-Dichloropropene and 1, 2-Dichloropropane) applied at29.6 31 Chapter 2 Literature Review
L/ha controlled P. thornei and increased grain yield in Australia (Doyle et al., 1987).
However, Thorne (1961) believed soil fumigation was impractical due to the distribution
of P. thontei inthe soil profile.
The most effective control in Australia is found with the granular carbamate Aldicarb
(Temik@) (Thompson et aL,1980a, b,1982,1983, 1984; Doyle et al., 1987 Klein er
al., 1987), increasing wheat yields by 1.6 tonnes/hectare or I3OVo in Queensland.
Application of 10kg Aldica¡b /tra before the planting rain, worked into the soil the day of
planting, or with 2-5kg Aldicarb /tra applied as granules in the seed row, provide the best
results. However, this is uneconomic in field situations, and the best results obtained at
any rate of Aldicarb was for the value of the extra grain produced to just match the cost of
the nematicide (Thompson ¿/ al., 1982). Other carbamates (Oxamyl, Carbosulfan or
Cleothocarb) and organophosphates (Fenamiphos, Terbufos, Ethoprop and
Fensulfothion) were less effective than Aldicarb (Thompson, private cornm. 1993).
Further studies conducted in Queensland indicated coating seeds with the carbamates,
Carbofuran and Oxamyl (0.125-0.5 kg/tra) resulted in only a slight reduction in numbers
of P. thornei and had little effect on yield (Thompson, private comm. 1993). However,
in Israel Furathiocarb (lOg/kg seed) or Oxamyl (3.6g/kg seed) reduced P.mediterraneus populations by 65-80Vo and increased ear count and grain yield by ZO-3IVo and 48Vo respectively (Orion and Shlevin, 1989), and were economically viable for use on low cash crops grown in marginal areas. However, in most cases nematicides are not an economically viable proposition for P. thoruei control in cereals (Van Gundy et aI., 1974;
Thompson, private coÍrm. 1993). 32 Chapter 2 Literature Review
2.9.2 Cultural Practices
Cultural practices imply the use of crop rotations, tillage practices, soil solarisation and time of planting. DiVito et aI. (1991) found that mulching fields with polyethylene film for up to 8 weeks suppressed populations of P. thornei by 50Vo. The reduction in nematode numbers was correlated with an increase in grain yield for plots treated for 6-8 weeks. It may also be possible to control P. thornei by manipulation of planting dates to avoid peak activity of the nematode. Van Gundy et al. (1974) found delaying sowing of winter irrigated wheat by 1 month in Mexico gave maximum yields. However, in NSW
Pattison (1993) suggested that wheat crops should be sown early to allow maximum root development to occur when temperatures are less favourable for P. thornei multiplication
(April and May).
In Israel, Orion et al. (1984) found that biannual fallowing reduced P. mediteraneus populationsby 90Vo and increased grain yield by 40-90Vo. In Mexico the numbers of P. thornei were higher in wheat-fallow-wheat rotations than those involving Zea mays,
Gossypium spp. or Glycine max (Yan Gundy et al., 1974). The lowest numbers of P. thornei were associated with rotations which were out of wheat for two consecutive years. The host range of P. thornei is wide (see section 2.3), with 18 botanical families
being affected. Triticum aestivum and Phaseolus lunatus are good hosts of P. thornei , fair hosts include Zea mays, Secale cereale, Glycine max, Hordeum vulgare and Avena sativa, while Sorghum vulgare is a poor host (Van Gundy et aI., 1974). Both
Thompson and Clewett (1986) and Van Gundy et al. (1974) suggested that fields infested with P. thornei should never be sown with wheat in succession.
Rotations involving crops other than wheat which favour lower nematode multiplication can be devised. Thompson et aI.(1981) suggested that Clipper barley was partially resistant to P. thornei and could be grown in place of the second wheat crop after a long 33 Chapter 2 Literature Review fallow on the Darling Downs in Queensland. Another common practice in Queensland is to alternate wheat with sorghum accompanied with long fallows. Experience on the
Darling Downs suggests that current rotations from wheat to non-host crops of setaria, linseed and canary and the inefficient hosts of sorghum, sunflower and pigeon pea, are suitable to keep P. thornei at non damaging levels, provided wheat is grown no more frequently than once every three years (Clewett et a\.,1993). Diversification of cropping patterns to include susceptible crops like chickpea, mungbeans, triticale, muze and barley leave moderate to high residual populations of P. thornei and provide less satisfactory breaks for wheat in nematode infested fields (Clewett et a1.,1993). In NSW, yield of barley and sorghum was satisfactory with the yield of the next wheat crop improved, however the subsequent wheat crops yielded poorly (Doyle et aL.,1987). Esmenjaud et al. (1990) showed that P. thornei, P. neglectus and P. crenatus were most numerous on wheat after maiza with wheat monocultures having intermediate numbers. Inigated sugarbeet decreased populations of Pratylenchus, with the effect sustained even on the third crop after sugar beet. In Utah, rotations with alfalfa and sugar beet for several years reduced P. thornei populations to negligible numbers (Thorne, 1961).
Oostenbrink et aI. (1956), found that flax, peas, potatoes and beet reduced P. thornei populations in Holland.
An eleven year management trial conducted on the Darling Downs at the Hermitage
Research Station revealed that the top soil of zero tillage fallow systems had higher P. thornei populations than mechanically cultivated treatments (Thompson ¿t al., 1983).
Further studies in Queensland revealed an increase in P. thornei numbers throughout the soil profile where stubble was retained for two years. Minimum soil disturbance in wheat fields favoured high numbers of root lesion nematodes (Klein et a1.,1987). In a four year continuous wheat trial on a red-brown earth, on the Darling Downs, 107 P. neglectus 12009 topsoil were found from zero tillage plots, but only 15/2009 from mechanically cultivated plots (Thompson, private comm. 1993). S. Taylor's (pers. 34 Chapter 2 Literature Review comm.) studies found the numbers of P. neglectus in Wallaroo oats were 557o lower in mechanically cultivated plots compared to reduced tillage treatments attributing Ìo a27%o increase in yield with cultivation. However, Overnoff (1991) found P. neglectus densities were greater in conventional than in no-tillage systems.
Organic amendments to the soil may also offer some control for P. thornei. Soil samples taken at Hermitage Research Station after 18 months of a weed free fallow showed significantly fewer P. thornei with stubble retention than with stubble burning (Thompson, private comm. 1993). Esmenjaud et aI. (1990) found that wheat monoculture with straw ploughed into the soil supported significantly fewer Pratylenchus than when straw was removed.
2.9.3 Biological Control
Successful biological control of Pratylenchus species is likely to be difficult due to their migratory behaviour. Pratylenchøs spend much of their lives in roots and tend to be
found in soil only when their host plants are stressed, senescing or diseased, or when their hosts have been ploughed out after harvest (Stirling, 1991). Since most eggs are laid in the root tissues and juveniles can hatch and develop to maturity without moving
from roots, multiplication sometimes can proceed for several generations without
nematodes being exposed to soil-borne antagonists (Stirling,I99I). Antagonists with
some specificity towards the target nematode are required. The bacterial parasite
Pasteuria thornei is a proven pathogen of P. brachyurus but has not been fully tested
against other Pratylenchus species (Starr and Sayre, 1988). Although the bacterium has been found in Australia nothing is known of its role in nematode population dynamics
and epidemiology (Stirling, 199 1). 35 Chapter 2 Literature Review
The fungus Hirsutella rhossiliensis which produces adhesive conidia was virulent to P. penetrans on potatoes (Timper and Brodie, 1993). The trapping fungi Arthrobotrys dactyloides, A. oligospora, Monacrosporium ellipsosporum and M. cronapagum trapped and killed most P. penetrans added to fungal cultures (Timper and Brodie , 1993). Pria et aL (1992) had similar findings in Brazil in aseptic Petri dish experiments with a selection of adhesive fungi (Arthrobotrys musiformis, A. oligospora, A. oviformis,
Monacrosporium eudermatum and M. gephyropagum) and adhesive knob fungi (M. ellipsosporum, M. parvicollis, M. drechsleri and Monacrosporium sp.), where all of the fungi where found to be highly predacious to Pratylenchus sp.. In Australia, field observations by Thompson ¿/ al. (I98Oa) suggested that the nematode trapping fungus,
Arthrobotrys conoides may limit populations of P. thornei. Other work by Gapasin
(1986) in the Philippines found that the fungus Paecilomyces lilacinøs, which is considered to act as an egg parasite (Stirling, 1991) reduced Pratylenchrs sp. on corn.
2.9.4 Resistance and Tolerance
A plant is considered resistant when the ability of the nematode to feed, develop and reproduce is inhibited, but if reproduction of the nematode occurs the plant is said to be susceptible (Wallace, 1963). A plant which is infested by a high number of actively reproducing nematodes but shows little indication of injury is tolerant (V/allace, 1963).
A tolerant plant is advantageous for growth and yield, but is generally associated with a high population of the nematode in the soil to infect the next susceptible crop. Breeding resistant crop varieties is therefore a priority area for the control of P. thornei. Such varieties would result in äecreased nematode populations which would allow increased yield and would also lessen the chance of spreading the infestation to unaffected soil
(Anon., 1990). Farmers would also have the benefits of more flexibility in choice of
cropping sequences and control without increased production costs. 36 Chapter 2 Literature Review
Van Gundy et aL(I974) tested 51 different varieties and selections for resistance and tolerance to P. thornei. All varieties and selections tested were susceptible to invasion and reproduction of P. thornei, but some showed tolerance to P. thornei. Thompson and
Clewett (1986) assessed the tolerance of wheat cultivars using Aldicarb (5kg/ha) as a control, and identified genotypes with greater tolerance than any of the recommended varieties. The three barley varieties recoÍrmended for Queensland have more tolerance than the recommended wheats for this region. A range of tolerances to P. thornei extst among wheat cultivars which have been recommended for commercial sowings in
Queensland. Gatcher is highly intolerant while Gamut, Hartog, Oxley and Cook are cultivars with better P. thornei tolerance.
O'Brien (1983) was unable to detect any P. thornei resistance of wheat lines in pots.
However, there are several wheat lines with superior tolerance and partial resistance.
Thompson and Clewett (1986) have identified possible resistance and tolerance from the commercial Queensland wheat varieties. The intolerant variety Gatcher was found to produce 1 plant in about every 3000 which was highly tolerant and also had some resistance (Thompson and Clewett, 1986). One selection (GS28) has outyielded all commercial wheats on nematode infested soils and although resistant to stem rust, is susceptible to leaf and stripe rust. Another wheat selection, QT2997 (Potam/Cook) had superior tolerance but was susceptible to a new strain of stem rust. Recently a stem rust resistant selection QT4118 (Pedigree Potam 70l4*Cook) from QT2997 was released as the new variety "Pelsart" which offers superior tolerance, but no resistance to P. thornei
(P. Brennan, pers. comm.).
Another current program in Queensland is the screening of known tolerant wheats for resistance. At present there are no coÍrmercial wheat varieties available in Queensland with P. thornei resistance, although the line GS50A selected from the highly susceptible 37 Chapter2 Literature Review
Gatcher has been identified (J. Thompson, pers. comm.). Field tests enabling quantitative comparisons of genotypes at a range of initial P. thornei populations are being assessed to determine which cultivars combine superior tolerances with a high level of partial resistance. Thompson suggests that wheats with least tolerance in field trials are those which showed the highest P. thornei multiplication rates in O'Brien's studies
(1983). It has also been suggested that high populations of P. thornei will limit their own further multiplication on susceptible wheat cultivars of low tolerance (Thompson, private comm. 1993), but would suffer a yield penalty. Results with intolerant cultivars such as Gatcher suggest that growing these varieties as a second wheat crop resulted in fewer P. thornei and more residual soil nitrate and water for the third wheat crop which yielded better than the second. Interestingly, wheat cultivars with resistance to Cereal
Cyst Nematode, H. avenae differ in resistance to P. thornei. Growth of H. avenae resistant AUS10894 resulted in moderately high numbers of P. thorn¿¡, whereas1L avenae resistant Festiguay and 1L avenae susceptible Halberd and Egret led to low numbers of P. thornei (Thompson, private conìm. 1993). Alternation of H. avenae resistant and susceptible cultivars is a possible way of exploiting the inverse relationship between P. neglectøs and Cereal Cyst Nematode, whilst controlling cyst nematode populations in intensive cereal producing systems (Lasserre et aI.,1994).
In South Australia, resistance and tolerance to P. thornei has yet to be adequately assessed. Until such information is obtained, the cultural practices of rotations and cultivation offer some means of partial control. 38 Chapter 3 G eneral Laboratory Tec hniq ue s
Chapter 3 General La 3.1 Nematode Inocula
Inocula of P . thornei were obtained from carrot cultures (Plate 3 . 1 ). This method of culturing was modified by Nicol (1991) after Moody et al. (1973). Originally, the carrots were inoculated with P. thornei isolated from Urrbrae loam at the Waite
Agricultural Research Institute by Mrs. Heather Fraser, in 1991. Carrot cultures were inoculated using either sterile nematodes, individual pieces of already cultured carrot or with P. thornei reared on chickpea callus on White's medium (Nicol & Vanstone, 1993).
Cultures were kept at 2O"C in the dark for a period of between 2-3 months after which time more than 200,000 P.thorn¿i could be extracted (Plate 3.1). Evidence of sufficient population development was provided by the cow-web like expression of nematodes on the interior walls of tubs. If cultures were left too long, the structural integrity of the carrot collapsed and the culture was unusable (Plate 3.2).
P. thornei were extracted from carrot cultures by cutting carrots into 0.5cm slices in a laminar flow cabinet and placing the slices in petri dishes with SDW covering the cut section. The nematodes migrated from the carrot medium into the surrounding water over a period of several hours. The water was then passed through a sintered glass filter
(pore size 15pm) to reduce.volume. Subsequent dilutions were made to obtain the desired density of P. thornei.
Experiments which involved the closely related species P. neglectøs were similarly prepared from carrot culture, originally maintained by Dr. V.A. Vanstone at the Waite
Agricultural Research Institute. The original source of P. neglectus was wheat roots collected from the South Australian cropping region, Palmer (V. Vanstone, pers. comm.). Chapter 3 General Lab oratom Te c hniq ue s
Plate 3.1 : Representative replicate of the P. thornei caffot culturing technique in tubs 12 weeks after inoculation with l0 P. thornei per carrot piece. Note the cobweb expression of. P. thorn¿i on the interior of the tub walls.
Plate 3.2 : Illustration of the various stages of development of P. thornei using the carrot culturing technique. Left; reduction in the size of carrot accompanied by some evidence of cell proliferation on the exterior portions of the carrot. Middle; the honeycomb effect caused by the swarming of nematodes, indicating harvesting is appropriate. Right; rapid blackening of carrot and deterioration in structure. The nematodes previously associated with the tub walls are collected on the bottom, in a rotten liquid mass in the base of the tub.
Chapter 3 Ge ne ral Lab orat om T e c hnio ue s
Plate 3.3 : The mister extraction funnel system used in the root extraction of nematodes. There are four funnels displayed, each having a coarse mesh disk wrapped in Kleenex @ tissue, fitted with a funnel below. Water is projected in a fine water spray and the nematodes are collected either in a test tube (as with the two left funnels) or in the bottom of a sealed tube (as with the two right funnels).
Plate 3.4 : Section of mister chamber used for the funnel extraction technique of nematodes within roots. Each rack has twelve holes to hold funnels (see Plate 3.3). The spray jets (shown at the top of the plate) are located above the racks within the chamber and project a fine water spray every 5 minutes for 20 seconds at 30 psi over the roots.
39 Chapter 3
G ene ral Laborato rv T e c hni a ue s
3.2 Nematode Extraction 3.2.1 Soil
Root lesion nematodes were extracted from soil (2009 composite sample) using the
Whitehead tray extraction technique (Southey, 1986). Two layers of Kleenex@ tissue were placed in a plastic basket (265 x 300mm) with the soil evenly distributed. The basket with soil was then placed in a V/hitehead tray (270 x 310mm) with 1500m1 tap water and extracted for 48 hours at room temperature. The tray with soil was gently removed and the water remaining in the Whitehead tray passed through a sintered glass filter (pore size 15pm) to a reduced volume of 20-30 ml. Nematodes in the sample were identified and then counted (Section 3.3).
3.2.2 Root
Nematodes were extracted from roots of various plants (cereal, legume or non-legumes) by misting (Southey, 1986). Roots were cut into 1.0 cm segments and spread evenly on a coarse mesh disk wrapped in Kleenex@ tissue which fitted into a funnel (Plate 3.3).
The funnels with disks were placed in a mister (Plate 3.4), which projected a fine water spray every 5 minutes for 20 seconds at 30 psi over the roots. The water and nematodes were collected in a test tube at the base of each funnel. After three days, the water containing the nematodes in the test tube was reduced using a sintered glass filter (pore size 15pm) under suction and the nematodes were then identified and counted (Section
3.3).
3.3 Nematode Counting
All nematodes, whether from soil or plant, were counted in a modified Doncaster (1962) counting dish (3cm diameter) with 4 concentric circles using a light microscope. A lrrl subsample from a known volume was used to estimate the numbers of P. thornei and P. neglectus where applicable. The 2 nematode species could only be distinguished in the adult stage by the position of the vulva in the female (Appendix D). P. thornei and P. neglectus in the juvenile stages were classified as Pratylenchus spp. 40 Chapter 3 General Laborato n Technia ues
3.4 Staining Nematodes and/or Fungi 3.4.1 Nematodes Only
Nematodes inside root tissues were stained with acid fuchsin (Southey, 1986) in lactoglycerol to detect larvae, adults and eggs of PraryIenchus. The modified method developed by A. Taheri (unpublished) was used to clarify nematode staining against root tissue. This involved soaking about ten root segments of approximately 10cm length in
20ml of chlorine bleach (4.47Vo NaOCI) for l0 minutes followed by rinsing for 60 seconds in SDW. The root segments were then allowed to soak for 15 minutes in SDW, drained and added to a3.5Vo solution of acid fuchsin (Southey, 1986), and boiled over a low flame for 30 seconds, and allowed to cool at room temperature. The roots were placed in 20ml glycerol with 10 drops of 5M HCI and heated to boiling. The pure glycerine and roots were then transferred to a petri dish and nematodes viewed microscopically.
3.4.2 Nematodes plus Fungi
Taheri (unpublished) developed a new technique for staining both nematodes and fungi, maintaining the resolution of both. The root segments were fixed in 4:1 FA (formalin: acetic acid ) fixative over night. The root segments were rinsed several times with SD'W, placed in 57o KOH and stored at room temperatures for 12-24 hours, depending on the age of the root system. The roots were then rinsed again with SDW and transferred to
O.OL Vo trypan blue in lactoglycerol for 1-2 hours at room temperature. They were rinsed and transferred into destaining solution (10 drops of HCI in 2O ml of glycerol ) for storage before microscopic examination. The hyphae, spores and nematodes were stained blue.
3.5 Seed Sterilisation and Germination
Seeds of cereals and non-leguminous hosts were selected for uniformity of size and washed in ethanol (98Vo) for 5 minutes, then surface sterilised with sodium hypochlorite 4I Chapter 3 G ene ral Labo ratory T e chni q ue s
(4.5Vo) for 10 minutes, with occasional agitation. The seeds were rinsed three times in
SDW, placed in plastic petri dishes (9cm diameter) containing sterile moistened filter paper (approximately 20 seeds/ petri dish ) and incubated at 20"C in the dark for 48 hours. Uniform seedlings of both cereal and non-legumes were selected on the basis of
3 roots of equal length (approximately 3cm).
3.6 Statistical Analysis 3.6.1 Statistical Designs
Depending on the aims of the experiment and the resources available various statistical designs were used (see for example Sokal and Rohlf, 1969). These designs included;
CRD (Completely Randomised Design) which can be used to compare several treatment classes or types simultaneously, where there is no indication of heterogeneity over the experimental units and the total number of experimental units (e.g. plots or tubes) is not large. The treatment classes or types are randomly allocated to the experimental units such that there is equal (or near equal) replication.
RCBD (Randomised Complete Block Design) is used where one wishes to remove the effect of heterogeneity over the total experimental units thereby increasing the precision associated with the comparison of the treatment classes or types. Each block contains as many experimental units as there are treatment classes to be compared with random allocation of the treatment classes to these units.
SPD (Split Plot Design) is used when there are two or more treatment factors and there are management or other reasons for randomly allocating one factor to sub-plots within whole plots of the second factor. Blocking may or may not be applied to the whole plots of the SPD and the concept can be extended to include more than two treatment factors if desired.
3.6.2 Analysis of Variance and Error Bars
All data were subjected to the appropriate analysis of variance (ANOVA) corresponding to the design used. This method, based on least squares, was pioneered by R. A.
Fisher, and is fundamental to much of the application of statistics in biology (Sokal and 42 Chapter 3
associated null hypothesis versus the alternative hypothesis (Moore and McCabe, 1989).
If the null hypothesis, that all treatment classes have the same mean is rejected then it is useful to present means, in a table or simple figure, and indicate which means differ significantly from others by including the Standard Error of Difference (SED) of Tukey's
Honestly Significant Difference or Range (Sokal and Rohlf, 1969). Other specific statistical tests are applied where appropriate with due reference given in the text. 3.6.3 Transformations
If there is evidence of failure of the assumptions underlying the analysis of variance the appropriate transformation is applied. Testing of assumptions is particularly achieved using plots of the residual values versus the fitted values from the ANOVA. The assumptions most frequently failing are, homogeneity of variance over the treatment classes, and normality of the residual variation (Atkinson, 1985). On the occasions when the assumptions fail either a square root 1r/ilõ3¡ or a logarithmic (log"(x+l.0)) often overcame the problem.
3.7 Classification of Soils used in Laboratory Experiments
The mechanical analysis using the hydrometer method described by Day (1965) was used to classify the three different soil compositions used in laboratory experiments involving the root lesion nematodes. The method principally involved the dispersion of soil particles over time after all aggregates were removed by chemical treatments, allowing the primary particles to be dispersed in water. In addition, the pH of the 3 soils was determined using 2 methods, 0.01M CaCl2and DDW (Rayment and Higginson, 1992).
The soil and pH classifications are presented in Table 3.1. Further explanation of soil texture is referred to in Table 4.1.
Table 3.1 Soil and pH classification of the soils used in Laboratory Experiments Vo Clay Vo Silt VoSand pH CaCl2) (DDw) Urrbrae loam (Ul) r4 25 6t 5.2 5.4
Roseworthy sand (rs) 4 1 95 7.r 8.2
Palmer sand (ps) 9 1 90 7.5 8.4 43 Chapter 4 Statewide Survey for Pratylenchus
Chapter 4 Statewide Survey for P. thornei and P. neglectus in the Cereal Regions of South Australia 4.1 Introduction
Pratylenchus thornei was first recorded in South Australia by J. Fisher in 1956 (pers. comm.). Since then it has been found in the Adelaide metropolitan area (Grandison,
1972; Singh, 1984) and in paddocks sampled across the cereal growing regions (S.
Taylor and V. Vanstone, pers. comm.). Commonly, P. thornei has been associated with the closely related species P. neglectøs (S. Taylor and V. Vanstone, pers. comm.).
However, the distribution of P. thornei and P. neglectus across the cereal regions of
South Australia has not been clearly defined.
In South Australia, P. thornei can cause significant yield reductions of wheat both in the field (Taylor and McKay, 1993) and in aseptic laboratory studies (Nicol, 1991).
Evidence from this work and the literature suggest P. thornei is usually found in clay 'Wallace, based soils, both in Australia (Grandison and 19741, Doyle et al., 1987) and overseas (Thorne, 196l; Loof, 1960; Corbett, 1970; Van Gundy et al., 1974 and
Fortuner, 1977).
In order to determine the damage potential of P. thorn¿i on wheat in South Australia, a study of the distribution of the two species, P. thornei and P. neglectus is essential. A statewide survey of the cereal growing regions was carried out involving the assessment of over three hundred soil samples accompanied with sampling over one hundred different types of commonly cultivated plants comprising different species and cultivars. The plants were taken from the same paddock as the respective soil samples.
Attempts were made to correlate the species of plant nematode with soil type. 44 Chapter 4 Statewide Survey for Pratylenchus
4.2 Materials and Methods
For the purposes of the survey, the cereal growing regions in South Australia were subdivided into nine geographical regions (Fig. 4.1). Within each subdivision both soil and root samples were taken from a wide cross section of the region concerned. The survey was carried out over a period of three consecutive years. Emphasis was placed on sampling clay-based soils due to the suggested association of clay soils and P. thornei (Fig.4.1).
The main objective of the survey was to determine the presence or absence of the nematode species, rather than precisely quantify the nematode population densities.
The large area of the survey limited the extent of indivudal samples taken from each paddock. As a consequence the information gained should be used in a qualitative rather than a quantitative sense.
A range of farms across the state were selected by contacting district agronomists,
Agricultural Bureaus and Land Care Groups. The individual farmers and paddocks were selected to give a cross section of the geographical area and crop rotational sequence.
From each individual paddock, soil and sometimes plant roots were collected.
Approximately 3009 of soil was collected to a depth of 20 cm across five sections of the paddock approximately 10m apart. The five soil samples collected from each paddock were combined and mixed to give a homogenous 1.5 kg sample. Where plant samples were taken, two or three plants were uprooted in each of the 10m transects where soil was obtained. Plant samþles were analysed immediately while soil samples were stored at 4"C and processed within a fortnight.
45 Chapter 4 Statewide Suney for Pra\lenchus
The soil type was classified as sand, loam or clay on the basis of texture in the field by the 'feel method' (Brady, 1984) and the soil terminology used is explained in Table 4.1.
This involved adding water to the field soil and once wet the way it 'slicked out' when pressed between the thumb and fingers indicated the amount of clay present. The slicker the wet soil the higher the clay content. Sand particles are gritty, while silt particles have a floury or talcum powder feel when dry, and are only slightly plastic or sticky when wet.
Table 4.1 : General Terms used in the classification of soil based on texture. (sourced from Brady (1984) and Foth (1978), International Soil Science Society Standards) common Name Texu¡re Basic soil Texrure Diameær (mm) No. partiõlevg Sandy Soil coarse sands 0.2 - 2.00 720 loamv sands Loamy Soil mod.-coarse sandy loam fine sandy loam 0.02 - 0.2 46,000 mod. v.fine sandy loam loam silt loam mod.-fine sandy clay loam silty clay loam clav loam Clay Soil fine sandy clay silty clay 0.002 - 0.02 5,776000 clay < 0.0002 90,260.853000
Plant samples were collected to obtain a diversity of varieties and/or cultivars of cereal, grain and pasture legumes, non-leguminous and a wide array of weed species. This gave preliminary information about the ability of plants to be hosts for Pratylenchus spp. The rotational sequence in each paddock was recorded.
The methodology for nematode extractions for both the soil and plant root samples are described in Section 3.2.1 46 Chapter 4 Statewide Survey for Pratvlenchus
4.3 Results
The raw data from the statewide survey are presented in Appendix A. Extensive detail of the individuat farmers, specific cultivars/varieties and crude estimates of nematode numbers are given for future reference. Table 4.2 summarises the distribution of P. thornei and P. neglectus by region while Table 4.3 relates nematode distribution to soil type.
Due to the lack of adult specimens, not all samples could be positively identified as P. thornei or P. neglectus. As a result only a certain percentage of soil samples in each region or soil type could be positively identified (column 4, Table 4.2 and Table 4.3).
The number of samples positively identified was used to calculate the next four columns in both Table 4.2 and Table 4.3. The last column of data in both Tables was determined irrespective of positive nematode species identification, hence it was calculated on the total number of samples.
Table 4.2 : Distribution of P. thornei and P. neglectøs in relation to cereal growing regions in Southern Australia.
q' bolh % w¡lh ore or Year of Tofal m. lvel! ntll Vo Vo or P.lhomei P.Mglcctus P.lhonei & bolh P.lhon¿i Region collællon samples idenÍn€d s P.lhon¿i P. lhonei g¡d P.ncßl¿clus only only P.ncgleclus & P.n ttcclus ¡nd/or P. n¿elcctvs 0 80 20 100 I rilestern E)re I 993 54 r00 0 Peninsula 100 0 l0{l 2 Central Eyre I 993 t2 100 0 0 Peninsula o 100 J Eastem Eyre 1993 42 t00 0 0 100 Peninsula '15 t4 8!¡ Lower r993 36 100 il 0
34 0 t9 5 I¡wer-Mid. 1992 107 52 2t 45 Norrh 0 73 6 Upper 1992 22 4l 67 22 II Nonh Yorke t994 30 9'l 3 0 l0 87 n
33 r00 8 Murray 1994 E 75 0 0 67 Mallæ 17 t2 9 South 1994 22 86 2t 26 t6 East 47 Chapter 4 Statewide Survey .for P ratylenc hus
Table 4.3 : Distribution of P. thornei and P. neglectøs in relation to soil type in the cereal areas of Southern Australia.
Ysr of Tol{l no. lvel! % null Vo 9o lnah 9Í' w¡lh ore or Soil Closifìcation mll€cl¡on umphs ¡dentil¡ed s P.thonc¡ or P.lhonei P.nqlectus P.thonci & bolh P.lhom.¡ P.lhonci P.n ghcrus only only P.ncglcctus & P.ncglcclus ¡nd snd/or P- ncPlectus Clay t992 (smdlcst porc siæ) I 993 97 66 37 34 l8 t7 I qq¿ Loam t992 (modcrrtc fnrc siæ) t993 t84 89 t2 l2 6t t6 88 t994 Sand t992 q) (hrgcst fx)rc sizc) t993 52 92 il 6 63 20 t994
In 1992, over one third of the total samples were collected from the Lower, Mid and
Uppèr North of the State because of the preponderance of clay based soils in this region. Nematodes in only half of these samples were positively identified due to the lack of adequate adult specimens, possibly due to the prolonged wet season. Of the samples which could be identified nematodes were either solely P. thornei or P. neglectus. In the Lower-Mid North 45Vo of the samples were P. thornei only and,34Vo were P. neglectus, while in the Upper-Mid North there were more P. thornei than P. neglectus that were positively identified, and once again there were no mixes of species within samples.
On Eyre Peninsula, l00%o of samples were positively identified. All samples from
Central and Eastern Eyre contained P. neglectus only. Both Western and Lower Eyre had a small population of mixes of both species while the majority of samples were solely P. neglectus. The samples taken from Regions 7, 8 and 9 (Yorke Peninsula,
Murray Mallee and the South-East of South Australia) taken in 1994 could be identified to species level. On the Yorke Peninsula 97Vo of samples contained Pratylenchø.s spp.
In the majority of samples (87Vo), both species were present, but in the remaining IOVo only P. neglectus was identified. 67Vo of the soil samples from the Murray Mallee 48 Chapter 4 Statewide Survey for Pratylenchus contained P. neglectus alone, while the remainder (33Vo) were mixes of both species.
In comparison, 37Vo of samples from the South-East (which extended across the
Victorian border) had mixed populations, 26Vo had P. thornei only and l6Vo had P. neglectus only.
From some of the 325 soil sample sites, root samples were also collected. There were over one hundred of these root samples, taken from a wide range of plant crops including cereals, legumes (pasture/grain), non-legumes as well as common weed species (Appendix A). The crops sampled and the nematode species and numbers corresponding to each crop are presented. The qualitative nature of the information presented in Appendix A, does not, unfortunately, allow ranking of the varieties/cultivars from the field due to the many variables cited. However, the results suggest that the majority of plants used in rotation with wheat, and common weeds, need to be considered potential hosts for both P. thornei and P. neglectus.
4.4 Discussion
Pattison's work (1993) in Northern NSW on P. thorn¿i in wheat concluded that the distribution of the nematode was more random in wheat fields at sowing than other nematode species in other crops. This suggested that the sampling technique used can be less intensive than those recommended for nematode populations which are aggregated. However, although nematode extractions and counts were obtained from a composite sample of at least five transects within each paddock, the numbers resulting only provide an indication of their presence or absence. To use them to quantify density could be misleading given the differing times of collection, varying management practices and the differing crop maturity in different samples. 49 Chapter 4 Statewide Survey for Prafilenchus
As indicated in Appendix A, both P. neglectus and P. thornei were present in a range of different crop plants, including cereal, grain and pasture legumes as well as weed species. This supports the polyphagous feeding habits of both P. thornei (Table 2.1) and P. neglectus (Anderson and Townshend, 1976). In order to confirm this hosting ability of the range of crop plants assessed an appropriate resistance assay needs to be developed.
The results presented in Table 4.2 rcgarding the distribution of Pratylenchus sp. in
South Australia indicate that approximately 9OVo of soils contain either P. thornei and./or P. neglectus. The Eyre Peninsula (encompassing Western, Central, Eastern and
Lower sub-divisions) has a 977o chance of Pratylenchus sp. being present, with the majority of samples containing P. neglectus. There were isolated pockets of mixed populations on Western and Lower Eyre Peninsula. Unexpectedly around Minnipa and
Streaky Bay mixed populations of P. thornei and P. neglectus were found where the soils were very sandy and calcareous. This does not support literature associating clay soil type with P. thornei.
As shown by the results, the species distribution between regions is variable. It appears that both species are present in all regions except the Eyre Peninsula, where only P. neglectus occurs. The distinction between proportions of species (P. thornei and/or P. neglectus ) in a given region or soil type may be a reflection of the samplin gyear and/or previous rotation and./or management practices. However, the higher proportion of P. thornei in the Lower to Mid-North and the South-East may reflect the higher clay content of the soils sampled (Table 4.3,Fig.4.1). It is also possible that other soil characteristics, such as soil pH may play an important role in species distribution. 50 Chapter 4 Statewide Suntey for Pratylenchus
The relation of soil type to species is summarised in Table 4.3. Irrespective of soil type, there was a 90Vo probability of finding Pratylenchrr spp. The work of Wallace
(1968) suggested that optimum conditions for movement of both P. thornei and P. neglectus would occur in sandy soils. The distribution of P. neglectøs appears to be associated with optimal nematode movement, as the higher proportion of P. neglectus populations were found in sand. However, P. thornei was found more commonly in clay based soil (Table 4.3) with most of these soils being found in Lower to Mid-North and the South-East (Fig. a.1, Appendix A). This suggests that factors other than movement determine the distribution of this species. About 20Vo of all samples had mixes of both species and were found across all types of soils, suggesting that soil type alone does not determine PraryIenchrs spp. distribution. It is probable that if more samples had been taken the proportion of mixed populations would have been greater.
O'Brien and Stirling (1991) noted that as the number of soil samples increased, the likelihood of finding nematodes which are present in low numbers also increased.
Therefore, P. thornei and P. neglectus is widespread in cereal growing regions. They show polyphagy on cultivated wheat , common weeds and crops grown in many rotational combinations in the cropping regions of South Australia. Because of this, the damage potential of P. thornel is high, but this is dependent on initial nematode density.
In order to assess the hosting status of the these crops commonly used in cereal production, a resistance screening procedure needs to be developed. 5l Chapter 5 Multiplication of P. thornei
Chapter 5 Multiplication of P. thornei and Development of a Resistance Assay for Cereal and Non-Leguminous Hosts
5.0 General Introduction
Species of Pratylenchus have a wide geographical distribution, and a great diversity of
plants act as hosts to different species within the genus. As discussed in Chapter 2, the
life cycle is dependent primarily on host and temperature. Because Pratylenchus are
migratory endoparasites, they are attracted to roots and invade in a non-random manner,
subsequently feeding on and reproducing in the cortex (Baxter and Blake, 1967). The relationship of Pratylenchus with its host is less complex than that of other plant parasitic nematodes such as Meloidogyne or Heterodera as no specialised feeding nurse cells develop inside root tissues (Townshend, 1990).
Little is known about the susceptibility or resistance (Section 2.9.4) of various wheat cultivars/varieties and other rotational crops to P. thornei. To examine these aspects it is necessary to determine the multiplication of the nematode on the various crop plants and to assess the effect of container size, soil type and time allowed for multiplication on the numbers of nematodes that result. It is beneficial to compare the multiplication of the two closely related species, P. thornei and P. neglectus , as these commonly occur as mixed populations in South Australia (Ch. 4). From such information it may be possible to design a resistance assay that will produce results in minimal time and with optimal use of resources. Such information produced in the laboratory will help in understanding multiplication and damage in the field.
In South Australia, the majority of wheat is grown in rotation with other cereal and leguminous species including grain and pasture legumes. Cereals grown in rotation with wheat include barley, triticale, rye, durum and oats. The diversification of the 52 Chapter 5 Multiplication of P. thornei cereal industry has seen alternative crops also being used in rotations, for example canola. This crop, however, is currently confined to the higher rainfall areas. The relative susceptibilities of the different rotational crops to Pratylenchus spp. will be a major determinant of the initial density of the nematodes in the succeeding crop.
5.1 Cpmparative Multiplication of P. thornei over time
5.1.1 Introduction
The approaches used in screening for resistance are determined by the cultivars and facilities available. In the laboratory a number of techniques have been used to screen for resistance to Pratylenchus spp., including paper towelling, silica sand or agar media in test tubes, petri dishes or jars (Townshend, 1990). In addition screening for resistance to Pratylenchus spp. may be undertaken in a greenhouse using multi-celled containers such as Styrofoam trays or plastic trays of plastic pipe containing infested soil and plants of interest. The multiplication rate of the nematodes may be assessed on the basis of the capacity of the nematode to penetrate roots, cause lesions and reproduce. It is imperative that a non-damaging initial inoculum is used in order not to affect the subsequent multiplication rate.
The multiplication of P. thornei on a range of cereals and non-leguminous hosts was assessed in two different experiments; one in large pots consisting of 6 replicates grown for a whole season, and the other in small tubes consisting of 10 replicates grown only for 2 months.
5.1.2 Materials and Methods
A range of wheat cultivars was selected from Queensland Wheat Research Institute,
Sydney University and South Australia. Other species of cereal and non-leguminous hosts were selected from South Australia (Table 5.1). In addition, two non-leguminous 53 Chapter 5 Muhinlication of P. thornei hosts, linseed and canola, were also assessed because of the diversification of the cereal industry into alternative crops, albeit on a relatively small scale and in confined geographical areas.
Urrbrae loam, collected from fields around the Waite Agricultural Research Institute
(Section 3.7), was autoclaved at 180oC for 24 hours, and allowed to cool. It was then mixed to a homogeneous state and was added to a) 180 pots, 25 cm in diameter and23 cm deep, with 4.5 cm of wood chips in the base to enable water filtration; and b) 300 tubes made from electrical conduit, 2.7 cm in diameter and 12.5 cm deep, filled with soil to approximately 7 /8 of the height of the tube. Two hundred seeds of each variety/cultivar (Tatle 5.1) were sterilised, germinated and selected (Section 3.5).
Twenty four of the selected seedlings of each variety/cultivar were chosen and four plants were planted in each of the pots. Within each pot, they were planted equidistant from each other. This gave six pots of each variety /cultivar. Selected seedlings were planted into the tubes (one seedling per tube), and approximately 2 cmlayer of soil was placed over each seedling.
Table 5.1, : Varieties/cultivars tested in the comparative multiplication over time. (SA; South Australian, SUN; Sydney University; QLD; Queensland University)
VARIETES/CULTIVARS Triticum aestivum Wheat SA; Halberd Festiguay, Spear, Machete, Warigal, Molineux, RAC589 SUN ; Sun 277, Sun 2894, Suneca QLD ; QT 41 18, Banks, Hartog, GS50A. 0T5648. Gatcher 'lrittcum durum I)urum wheat Kamilaroi, Yalla¡oi Secale cereale SA Rye 'Iriticum secale 'l'riticale Currency Avena sp. Oats Marloo, Echidna, Quaker 84-187, Potoroo Hordeum sp. Grimmett, Skiff, Galleon Brassica sp. Rapeseed(Canola Golden Rape Linum usitatissimum Linseed Glenelg 54 Chapter 5 Multiplication of P. thornei
P. thornei were extracted from carrot cultures (Section 3.1). One week after planting, 1 ml of aqueous solution containing 500 P. thornei was evenly distributed over the soil root interface of the four seedlings. One month later the plants were re-inoculated with
1 ml of aTOO P. thornei /pot inoculum. Thus total inoculum was l2O0 P. thornei per pot. Similarly, tube seedlings were inoculated with 400 P. thornei, in 1 ml aliquots one week after sowing. These inocula were considered a non-damaging initial density
(Nicol, 1991).
Pots were arranged as a randomised complete block design (RCBD). Each pot of the
30 varieties/cultivars was considered as a single replicate, and all pots of each variety/cultivar were randomised within each of 6 blocks. Plants were grown under natural climatic conditions of the normal growing season (Plate 5.1), outside in a wire- grid bird cage. Plants were watered with tap water whenever necessary.
The tubes were also arranged as a RCBD. The 30 tubes of each variety/cultivar were randomised within each of 10 blocks. The tubes were individually embedded in a tray of soil, containing a wire grid to support the tubes (Plate 5.2). Plants were watered with tap water whenever required, so that water was not a limiting factor. Plants were placed in a controlled temperature growth room at20"C, with a 10 hour daylI{ hour night, and a light intensity of 65p Einsteins from fluorescent light tubes.
Plants in pots were harvested after 5 months (equivalent to the end of the season in the field). A composite soil sample was obtained from eleven cores covering a uniform cross section of each pot. Each core was 20 cm deep and 1.5 cm wide, and included both roots and soil. Nematodes were extracted by the Whitehead tray method (Section
3.2.1). The number of nematodes extracted from each sub-sample was converted to P. 55 Chapter 5 Multiolication of P, thornei thornei per gram soil and extrapolated on a per plant basis for the 4 plants within each pot.
Plants in tubes were harvested after two months. Plants were washed out of the soil and carefully rinsed to avoid root loss. Nematodes were extracted over a period of 3 days using mister extraction (Section 3.2.1) and counted (Section 3.3). In addition, the root systems were visually assessed for the degree of root lesioning on a scale of 0 (no lesioning), 1 (low lesioning), 2 (moderate lesioning) and 3 (high lesioning).
5.1.3 Results
All data from both experiments were analysed separately as a RCBD. The analysis on the original data showed heterogeneity of variance which was removed using a logarithmic transformation (loge(x+1)). In both pots and tubes, there was a significant varietal effect, before and after logarithmic transformation (Table 5.2).
Table 5.2 : ANOVA from the pot and tube tests to compare the numbers of P. thornei on cereal and non-leguminous hosts. POT(log transfonned) TUBE (log transfonned) d.f. m.s. v.r. Prob. d.f. m.s. v.r. Prob. block 5 30.38 9 5.53 block,plot vanety 29 13.37 2.39 <0.001 29 22.91 22.17 <0.001 residual r45 s.60 261 1.034 Total 179 299
ln both cases the variance ratio (v.r.) indicates there was very strong evidence to reject the null hypothesis that all varieties had equal numbers of P. thornei. Thus we concluded there is a significant difference between the varieties. The varietal effect is illustrated in Figures 5.1 and 5.2. The Tukey Honestly Significant Difference Value, or Tukey's Range (o = 0.05) (Section 3.6) indicates the magnitude of the difference required between varieties before they can be considered statistically different. 56 Chapter 5 Multiplicqtion of P. thornei
The statistical analysis was extended with the help of orthogonal comparisons which
enabled comparisons both between and within species for the pot and tube experiments
(Table 5.3).
Table 5.3 : ANOVA using orthogonal comparisons to compare selected varietal combinations in both pot and tube experiments POTQog transformed) TUBE(log transformed) Prob. Prob. wheat vs. oats <0.001 <0.001 wheat vs. barley 0.176 <0.001 wheat vs. linseed & canola <0.001 <0.001 QLD, NSV/ wheat vs. SA wheat 0.280 0.319 bread wheat vs. durum wheat 0.003 <0.001
Figures 5.1 and 5.2 indicate similarities for the two experiments. Wheat varieties have
a significantly greater number of P. thornei than oats, barley (tube only), linseed and
canola. This is further supported by data in Table 5.3. Bread wheats have significantly
more P. thornei than durum wheat, and there is no significant difference in the number
of P. thornei when the origin of the wheat is considered.
The original ranking of root lesioning was also compared using the Friedman non- parametric test (Colquhoun, l97I) which revealed significant differences in the degree of root lesioning on the root systems of the varieties tested. Fig. 5.3 illustrates the degree of root lesioning with wheat showing much more damage than barley, triticale, rye, durum and oats, which showed little evidence of lesioning (Plates 5.3, 5.4, 5.5).
The two non-leguminous hosts, linseed and canola, had negligible lesioning (Plate 5.6).
Finally, a comparison of the ranking of the varieties based on the number of P. thornei in pots and tubes was made using the non-parametric Spearman's Rank Correlation Test
(Sokal and Rohlf, 1969). This indicated a significant t value of 5.680 (P<0.0001) which suggests that similar results can be obtained in a period of 2 months using small tubes as compared to 5 months using large pots. Given the reduced time needed to obtain 57 Chapter 5 Multinlication of P. thontei results the use of tubes can provide notable advantages when assessing the resistance of va¡ieties.
Fig. 5.1 : Multiplication of P. thornei on cereal and non-leguminous hosts after 5 months in large pots in the glasshouse. 7 Tukey - 5.11 6 ilil1il1 (i.e., means must differ by this magnitude on a log crl a 5 il¡u scale to be different.) o I I È 4 ililil 6)l EI ol J ilt I ll || ililil ,Êl ;l I I ll|| lilt ililil ào 2 Jo 1 ilr ||| || ililil
0 ¡t It lilt ililil þeåËËEË åËåt*g g g$ €ËË # tr$;rug; variety/cultivar
Fig. 5.2 : Multiplication of P. thornei on cereal and non-leguminous hosts after 2 months in small tubes at 20"C in a controlled growth room.
8 TukeY = 1.7 7 (i.e., means must differ by this magnitude on a log 6 I I (t scale to be different.) È 5 9l EI I ol 4 I :l I O.l J il I Þo J 2 il I I
1 ¡l'l ll 0 llI I þË Ë#E Ëg Eå; Ëå€ËË ËËËË ËåË# E.E#Ë variety/cultivar 58 Chapter 5 Multiplication of P. thornei
Fig 5.3 : Friedman's ranking of root lesioning for cereal and non-leguminous hosts grown in small tubes for 2 months at20"C in a controlled growth room.
25
bo I 820 tililililtililillillllllil ct) q) l< q9 15 llilllilill¡ Il]¡ It bo
J¿ I lllltililililllililil ¡iËto
c\, -u5 IlltlllIt O ¡r tJr 0 ||||t -åE jËË IË ;*gt Ë ËËË# ËË Ë !Ë ËE Ë åËË variety/cultivar
5.1.4 Discussion
These results provide strong evidence that P. thornei multiplication rates differ notably on the varieties/cultivars tested. The wheat cultivars tested were moderately to highly susceptible; the triticale, rye, barley, oats and durum were moderately susceptible; while the two non-leguminous hosts linseed and canola were least susceptible i.e. they appear to have some degree of resistance. Lawn and Sayre (1992), working in highland
Mexico, found significantly greater reproduction of P. thornei on bread wheat than on either durum or triticale. Thompson (private comm. 1993) also found that barley was associated with low nematode multiplication; in contrast, triticale encouraged P. thornei multiplication. Further supporting work by Thompson (private comm. 1993) on the
Darling Downs suggested that wheat was associated with many residual nematodes while linseed rù/as associated with very low numbers. Similarly, field work by Chapter 5 Multinlication of P. thornei
Plate 5.1 Experimental pot set-up used to assess the multiplication of P. thornei over a range of cereal and non leguminous hosts. Plants were grown for 5 months in a
RCBD under natural climatic conditions in a glass topped enclosure. Plants were watered whenever necessary with tap water.
Plate 5.2 Experimental tube set-up used to assess the multiplication of P. thornei over a range of cereal and non-leguminous hosts. Plants were grown in a controlled growth room for 2 months in small polyethylene tubes embedded in a tray of soil, with a wire grid to support tubes. Light was supplied with fluorescent light tubes (65p
Einsteins) and plants watered with tap water whenever necessary. r r .;f,+
-R
\T\\Iì -È:F , ---/ -
z'-
.t=-FË ì=
l'- ¿/
¡l ú
\ - ' 5'1"r* { ,"i¡ Sæ- Chapter 5 of P. thornei
Plate 5.3 Representative root systems of the wheat cultivar Festiguay (left) and the oat cultivar Potoroo (right) grown for 2 months in tubes at 20oC after inoculation with
40O P. thornei per plant. The wheat shows much greater evidence of brown cortical lesioning (high), particularly on seminal roots. In contrast the oat shows little evidence of nematode symptoms (low).
Plate 5.4 Representative root system of the wheat cultivar Machete (left) and the barley cultivar Galleon (righÐ grown for 2 months in tubes at 2O"C after inoculation with 400 P. thornei per plant. The wheat shows much greater evidence of brown cortical lesioning (high), particularly on seminal roots. In contrast the barley shows little evidence of nematode symptoms (tow). HIGH LOW Chapter 5 P. thornei
Plate 5.5 A representative root system of the triticale cultivar Currency grown for
2 months in tubes at 20"C after inoculation with 400 P. thornei per plant. There is some evidence of cortical darkening on seminals, however in comparison with the wheats (Plates 5.3 and 5.4) root lesion index is low.
Plate 5.6 Representative root system of the non leguminous host Glenelg linseed grownfor2monthsintubes at20"C afterinoculationwith400 P.thornei perplant.
There is little (low) evidence of lesioning by the nematode. Íy
CURRENCY LOW 59 Chapter 5 of P. thornei
Vanstone (1993) on the closely related species P. neglectu.ç in South Australia, found
that rye, triticale, barley and durum were more resistant to P. neglectus than the majority of wheat cultivars.
The evidence suggesting that canola (rapeseed) is partially resistant to P. thornei may
be associated with nematode suppression by glucosinolates which are sulphur
containing compounds produced in all parts of the plant (Sang et al., l9g4). The
breakdown of glucosinolates forms various iso-thiocynates which are known to have
fungal and bactericidal properties. Methyl iso-thiocyanate is a breakdown product of methyl sodium which is an effective nematicide (Lear, 1956). The work of Mojtahedi et al. (1991) found that amending Meloidogyne chitwoodi infested soil with chopped up
Jupiter canola reduced the population significantly more than did amendment with wheat roots.
The number of nematodes extracted from the tubes was greater than from pots (Figs 5.1 and 5.2). Possible explanations for this are associated with the different experimental techniques used. Although plants in the pots were grown for three months longer than those in tubes, the extraction efficiency of nematodes from roots and soil in the pots may not have been as high as that used for tubes, in which nematodes were extracted from the roots alone. In addition, given the relative size distinction between pots and tubes, penetration in a larger volume of soil medium could be less effective.
Multiplication is ultimately determined by the numbers of nematodes which actually penetrate the root.
The fact that statistically comparable results were derived from the two experiments suggests the assay to measure multiplication rate of P. thornei can be reduced effectively to two months. In addition to this, the ability to distinguish varietal 60 Chapter 5 Multiolication of P. thornei differences in the tube experiment is three times greater than with the pot experiments
(Figs. 5.1 and 5.2) this once again may be attributed to the size of the assay system. The smaller the soil volume the greater and more consistent penetration of the nematode would be, associated with less variability between individual replicates within a treatment. 61 Chapter 5 Multiplication of P. thornei
5.2 Relationship between P. thornei and P. neglectus 5.2.1 Introduction
As discussed in Chapter 4, P. thornei and P. neglectus occur quite commonly together
in field situations on a range of soil types and different hosts in South Australia. For
this reason, it is important to gain an understanding of the relative susceptibilities of
similar hosts to the two species. Given that both species are considered to be
potentially damaging in South Australia, control measures for both species will need to
be developed and implemented.
An examination of the multiplication of P. thornei and P. neglectøs on a range of cereals and non-leguminous hosts was carried out in collaboration with Research
Associate, Dr Vivien Vanstone, Dept. of Plant Sciences, University of Adelaide.
Several wheat varieties were selected from the Australian Winter Cereals Collection for their known resistance to Cereal Cyst Nematode (Heterodera avenae), another common root pathogen on cereals in South Australia. Both Pratylenchus species were assessed on the same range of host plants to allow comparisons to be made.
5.2.2 Materials and Methods
The plants to be tested (Table 5.4) were set up in a similar way to those in the previous section (5.1) as a RCBD with 10 blocks and 22 varieties randomly allocated to the 22 experimental units within each block. Two hundred seeds of each variety/cultivar were sterilised, germinated and selected (Section 3.5). One seedling was grown in each electrical conduit tubes (2.7 cm width x I2.5 cm height) with autoclaved palmer sand
(Section 3.7), which had been sterilised at 65'C for 45 minutes. One week after sowing, seedlings were inoculated with 400 P. thornei and 400 P. neglectus per plant in 1 ml aliquots. This was considered to be a non-damaging density as the subsequent 62 Chapter 5 Multiplication of P. thornei
multiplication rate was not affected (Nicol, 1991). The nematode inocula were
obtained from carrot cultures of the two respective species (section 3.1).
Plants were placed in a controlled temperature growth room at 20"C, 12 hour day and
night, provided by fluorescent light tubes (65¡r Einsteins). Nematodes were extracted
from plants harvested after two months using 3 day mister extraction technique
(Section 3.2.2) and counted (Section 3.3).
Table 5.4: Varieties/cultivars used for comparison of susceptibility to P. thornei and p. (SA; neglectus. South Australian, SUN; Sydney University; QLD; Queensland, AWCC; Australian Winrer Cereals VAI{.I1IIÈS/CI.] T'riticum aestivum Wheat SA; Machete, RAC 589 SUN; Sun 223A, Suneca QLD ; GS28; GS50A, Banks, Potam, Gatcher AWCC; AUS49l 8, 4US7869, AUS 10938, AUS 4930, AUS 7639, AUS 13807, AUS 10894 Rye SA Rye Triticale Cunency, Tatiara Avena sp. Oats Marloo, Wallaroo Brassica sp. Rapeseed(Canola) Barossa
The range of cereal hosts was selected to provide a wide cross section of different wheat genotypes from breeding programs. The Queensland wheat variety GS50A is a resistant selection from the highly susceptible and intolerant wheat cultivar, Gatcher, while the varieties GS28, Banks and Potam are known to be tolerant, but linle is known about their susceptibility. The AV/cc selections, AUS10g3g, AUS4930, AUS13g07 and AUS10894, were obtained from Ms. Franky Green (SARDI) and investigated because of their known single gene resistance to H. avenae. The rye, triticale, oats and non-legume canola were chosen to see if previous results could be verified for both species of Pratylenchus. 63 Chapter 5 of P. thornei
5.2.3 Results
All data for both P. neglectu,s and P. thornei per plant were analysed separately as a
RCBD. The original analysis showed heterogeneity of variance, so the data was log transformed Logs(x+l). A significant varietal effect was found from the analysis of
variance for both P. thornei and P. neglectus, before and after log transformation
(Table 5.5).
Table 5.5 : ANOVA of P. thornei and P. neglectus on cereal and non-leguminous hosts.
log (lu. neglectus + l) Prob d.f. m.s. v.r. Prob. block 9 1.963 9 1.3355 block.olot stratum vanety 2t t7.r72 13.93 <0.001 21 1.5506 6.68 <0.001 residual t7s(r4) 1.232 188(l) 0.2323 Total 205/14 218(1)
The significant varietal effect is illustrated in Figure 5.4 andFigure 5.5, with the Tukey
Honestly Significant Difference Value (a = 0.05) indicating the range required to significantly differentiate between varieties .
Further partitioning of the variety mean square with orthogonal comparisons allowed selective comparison of varieties from both P. thornei and P. neglectus are summarised in Table 5.6.
Table 5.6 : Orthogonal comparisons comparing selective combinations of varieties for both P. thornei and P. neglectus. _ P. thomei (log transformed) P. neglectus (log transformed) Prob. hob. wheat vs' oats <0.001 <0.001 wheat vs'barley <0.001 <0.001 QLD, NSW whears vs'SA wheat 0.380 0.911 AWCC wheat vs'SA wheat <0.003 0.311 wheat vs' triticale 0.924 0.269 64 Chapter 5 of P, thornei
Fig. 5.4 : Multiplication of P. neglectøs on cereal and non-leguminous hosts after 2 months in small tubes of 20"C in a controlled growth room 9 TukeY 0.76 8 I t I t I I T I I T I I = (i.e., means must differ by this e1 I t I ilil il ililil il magnitude on a log scale to be different) il ll il il ililil ll lililt il il ilililil lilt lt ililil il Iililt lt il ilil ilt ll il il ililil ilililil il il il ilil ilr il ililil ilililil il il bo 3z il ilil ilt lt ililil lllililt il il I il ilil ilt il ll ilil ilililil il il 0 il lil ilt ililil 11 ililil il
æJ r l-..+ 6) c\¡i: oOo\: oñË:=ç(r:^ +ooæ x Ë¡rsjÆËåãËËäuËää Éta(hu)ä J-Éì5¿ v) -<-< variety/cultivar
Fig. 5.5 : Multiplication of P. thornei on cereal and non-leguminous hosts after 2 months in small tubes of 20oC in a controlled growth room. 9 TukeY 1.76 8 lll = (i.e., means ilr T I must differ by this E7 magnitude on a log scale to be different) I T t il il il I t ilililil il il il I ilil lt ilililil I il il il lilt il ilililil il il o )2 il il ilililil il il
1 il il il ilililil il il 0 il il ilililil il il (É
(ú u L) åÉã ËäsËi E ËË ËË ËË ËËã ã ã 65 Chapter 5 Multiplication of P. thornei
From the results presented in Table 5.6 P. thornei and P. neglectus behaved similarly, both having significantly higher numbers of nematodes in wheat compared to oats and barley. However, there was no significant difference between wheat and triticale varieties. The wheats obtained from different states in Australia, were not found to differ significantly for either Pratylenchøs species. However, the AWCC wheat lines had significantly different P. thornei multiplication.
The correlation between the multiplication of the two species P. thornei and P. neglectus was calculated using the non-parametrical Spearman's Rank test (Sokal and
Rohlf, 1969). A t-value of 2.086 was found, which was non-significant at the 5 Vo level. This implies that the multiplication rates of P. thornei and P. neglectus were not dissimilar over the range of hosts tested. This is evident from Figs. 5.4 and 5.5, where for example, the AWCC variety AUS4930 is ranked the 8th most susceptible variety for P. neglectus, but is the 4th least susceptible for P. thornei. Similarly, GS50A, (the variety selected by Queensland breeders for resistance fo P. thornei ) is the 10th most susceptible variety for P. neglectus, but the 3rd least susceptible variety for P. thornei.
In contrast, Canola was the least susceptible of all varieties/cultivars tested irrespective of P ratylenclrøs species.
5.2.4 Discussion
The results suggest that there is a range of susceptibility to both P. thornei and P. neglectus within the cereals and non-leguminous hosts tested. The multiplication rate of P. neglectus was much greater for all varieties/cultivars tested than for P. thornei
(Figs 5.4 and 5.5). The statistical distinction between varieties for P. neglectus was limited by the similar range of susceptibilities (7.2 to 8.8 on the logarithmic scale) and the range required for distinction (Tukey = 0.76). The range of susceptibilities for P. thornei was much greater (2 to I on the logarithmic scale), but the range for distinction 66 Chapter 5 P. thornei was two fold greater (Tukey = I.76). Nevertheless, P. thorn¿i offers a much greater range of susceptibilities for breeders to work with compared to P. neglectus. The wheats appear to show greater susceptibilities than the rye, oat and canola, supporting previous observations for both P. thornei (Thompson, private comm. 1993) and P. ne glectus (Vanstone, 1993).
A plant is termed resistant if it restricts or prevents nematode multiplication (Trudgill,
1991). From the data presented in Figs 5.4 and 5.5, a plant could be considered resistant if 400 or less nematodes per plant were recovered two months post inoculation
(ie. when the initial density or less was recovered). On the logarithmic scale 6 is approximately equivalent to 400 nematodes per plant. From Figure 5.4 we see that for
P. neglectus none of the hosts tested had nematode numbers at or below this level. In comparison, Figure 5.5 indicates that almost half of the varieties tested showed final P. thornei numbers that were less than 400. Caution should be taken in defining the critical cut-off for what may be termed a resistant host as it is unlikely that the initial inoculation density reflects the number of nematodes which penetrated the root. In fact, depending on the experimental conditions (soil type, host, temperature), the number of nematodes which initially penetrated the host could vary widely, but should, undoubtedly, be less than 400. The work of Phillips (1984) on screeningfor G. pallida resistance with potatoes also found that ranking clones was more reliable than absolute values for similar reasons.
The data presented suggest that within the cereals assessed there is a differential host status for the two nematode species, P. thornei and P. neglectus. In the event that both species occur in the field and require effective control with the use of resistant cereals, the probability that one cereal will provide this for both nematode species appears unlikely. The resistance of the variety GS50A, originally a re-selection from the highly 67 Chapter 5 Multiplication of P. thornei intolerant susceptible cultivar Gatcher (Thompson, private coÍrm. 1993), is evident for the South Australian P. thornei, but it appears highly susceptible to P. neglectus. In addition, a variety of wheat (AUS 10894) recently released in Victoria on the basis of field resistance to H. avenae is one of the most susceptible varieties for both nematodes. However, another AV/CC variety (designated 4US4930, originally from
Iraq) offered promising resistance for P. thornei, and also carries with it a single gene for H. avenae resistance (F. Green, pers. comm.). This is not suprising asTinline et al.
(1989) suggests that regions where crop plants evolved from wild progenitors, the so called centres of origin or gene centres, are rich in plant diversity and may afford good sources ofresistance. 68 Chapter 5 Multiplication of P. thornei
5.3 Development of a Resistance Assay
5.3.1 Introduction
The previous two sections showed differences in multiplication of P. thornei in different cereal varieties under different conditions and suggested that this could form the basis for an assessment of comparative resistance between varieties. To be useful, such an assay should be as quick as possible, use the optimum number of nematodes that produce adequate separation of resistant and susceptible plants and occupy as little space as possible in the growth room or glasshouse. These factors are examined in this section.
5.3.2 Materials and Methods
Six cereal varieties ranging from resistant to highly susceptible, 2 soil types, 1 container
size,3 nematode densities and 3 harvest times were investigated.
The 6 cereal varieties were chosen deliberately to express arange of susceptibilities to
P. thornei (Table 5.7), as previously displayed in Section 5.1 and 5.2.
Over one hundred seeds of each variety (Table 5.7) were sterilised, germinated and selected (Section 3.5). Two soil types were used. Urrbrae loam (clay based) from soil collected near the Waite Agricultural Research Institute (Section 3.7) was autoclaved at lz0oc and allowed to cool. The second type was a sandy soil from Palmer in South
Australia, which was heat treated at 65oC for 45 minutes. Both soil types were mixed thoroughly and sieved through a2mmmesh to remove organic matter and small rocks.
Large electrical conduit tubes (3.7 cm x 12.5 cm) were filled to approximately 7/8 th of the tubes' height with either Palmer soil or Urrbrae loam and 108 seedlings of each variety were sown, half in each soil type, with 1 seedling per tube, and then covered 69 Clupter 5 Muhiplication of P. îhornei
with about 2 cm of the appropriate soil type. P. thornei was obtained from carrot
cultures (Section 3. l).
Table 5.7 : Varieties used for comparison of susceptibility to P. thornei in the resistance assay. (SA; South Australian; QLD; Queensland University; AWCC; Australian Vy'inter Cereals Collection)
SPECIES TÐ P. tlnrnei
Triticum aestivum SA;Machete - highly susceptible QLD;GS50A-resistant AUS Tritico secale Triticale SA; Avena sativa lJats SA; Wallaroo - resistant Hordeum Barley SA; Galleon - moderately resistant vulgare
Three P. thornei densities were used in this experiment. 100, 200 and 400 P.
thorneilplant were added to seedlings one week after transplantation. These were
considered to be non-damaging initial inocula (Nicol, 1991).
The plants were aranged as a split plot design (SPD) with 6 blocks where each block
corresponded to a particular harvest time, and 6 whole plots within each block in which the 6 varieties were randomised. Within each whole plot there was a factorial anangement of the 2 soil types and 3 P. thornei densities. The tubes were placed in a wire grid in a centralised position in order to avoid border effects throughout the experiment and shading effects at the latter stages of growth. The wire grid was placed on a tray of sterilised potting soil (Plates 5.2). The tubes were firmly placed in the soil at the bottom of the tray. Plants were keep moist at all times.
Plants were grown in a controlled temperature growth room at 2l"C,12 hour day, 12 hour night with light intensity 300-400p Einsteins supplied with high pressure sodium 70 Chapter 5 Multiplication of P. thornei lamps and supplemented by fluorescent light tubes. The plants were sprayed with
Dichlorvos-l 14 one month after inoculation due to a mite infestation. There was also evidence of powdery mildew infestation which was combated with the routine use of
Bayleton (1 ml/L).
After I and 2 months the tubes were harvested and carefully rinsed in water to avoid root loss. Nematodes were extracted over three days using the Mister Extraction
(Section 3.2.1) and counted (Section 3.3).
Plants were healthy after the first month, but by the end of the second month they were suffering an unknown problem. This was possibly a nutrient deficiency, toxicity or possibly another disease. Plants were not harvested after the third month as the results from the first two months indicated great variability, and the plants were suffering from head sterility and chlorosis by the third month.
5.3.3 Results
The original data showed heterogeneity of variance so a logarithmic transformation was applied (logs(x+l)). The results were analysed separately for each harvest, rather than including harvest time as an additional factor.
At both harvests, the number of P. thorneilplant significantly increased with initial nematode density (Fig. 5.6). Fewer nematodes were within the roots at harvest I than at harvest 2, at all densities. At harvest 1, only 5.5 - 6.5Vo of the inoculum had penetrated the root system, irrespective of initial density. At harvest 2, after some multiplication the percentage of nematodes within the roots compared with the initial density had risen to 17.0 - 22.5Vo for the 3 inoculum levels used. 7t Chapter 5 Multiolication of P. thornei
Table 5.8 : ANOVA for Harvest 1 and Harvest 2 to compare the numbers of P. thornei on cereal and non-leguminous hosts.
HARVEST I Qog transformed) HARVEST 2 (log t¡ansformed) d.f. m.s. v.r d.f. m.s. v.r. Prob. block block.plot.subplot 5 t.736 5 3.083 vanety 5 1.736 2.t4 0.093 5 38.383 8.43 <0.001 residual 25 52.39 25 4,526 block.plot.subplot soil I 0,063 0.03 0.860 I 3.556 r.32 0.2s2 nem.density 2 38.61 19.26 <0.001 2 25.51 9.50 <0.001 variety.soil 5 2.365 1.18 0.322 5 6.661 2.48 0.035 variety.nem density 10 1.936 0.97 0.4't6 10 2.567 0.96 0.485 soil .nem density 2 0.011 0.01 0994 2 r0.95 4.08 0.019 variety.soil.nem density l0 2.522 t.26 0.260 10 2.283 0.85 0.581 Residual 143(7) 2.00s 142(8) 2.68s
Total 208(',7) 207(8)
Fig. 5.6: The effect of P. thorn¿i initial density on the number of nematodes extracted after one and two months in large tubes at 20"C in a controlled growth room.
4.5
4.0 I 100 P. thomeilplant SED=0.27 3.5 S 200 P. thorneilplany SED=0.23 3.0 E 400 P. thomei./planr -o. sED=0.24 EI 2.5 rlbt dl 2.0 èo o J 1.5
1.0
0.5
0.0
harvest 1 harvest 2 72 Chapter 5 of P. thornei
Fig. 5.7: The varietal effect on the number of nematodes extracted after 2 months in large and small tubes at 20"C in a controlled growth room. (The data for small tubes was sourced from the same varieties from Section 5.1 (Ch 5), with the exception of Galleon which was not used in that experiment).
I
7 I Section 5.3 (current) SED=0.34 b $ Section5.l SED=O.45 (d 5 E. ol Él 4 EI -çl ;l 3 à0 Io 2
0 Wallaroo Galleon GS50A AUS4930 Mach€te Currency
variety
Fig. 5.8 : The interaction between soil type and variety on the number of P. thornei per plant after 2 months in large tubes at 20"C in a controlled growth room.
6.0
5.5
5.0 sana 4.5 I E Uubrae loam ã ¿.0 Ë sED<).63 'El s.s FI Ël s.o il ,, Þ9 J 2.o
1.5
1.0
0.5
0.0 Galleon Wallaroo AU54930 GS50A Machete Currency variety "t3 Chapter 5 ofP thornei
Fig. 5.9 : The interaction between nematode density and soil type on the number of P thornei per plant after 2 months in large tubes at 20'C in a controlled growth room.
5.0
4.5 I 100 P.thornei/plant 4.0 E zoo!4i/plant N 4ooL!@91/plant Ë 3.s ã(q SED=0.39 Èr 3'o EI €l 2.s ;l s 2'o 1.5
1.0
0.5
0.0 sand Urrbrae loam
soil type
At harvest 2, "varíety" had a significant effect on numbers of nematodes in the root systems (Fig 5.7), with Machete wheat having the most nematodes followed by the triticale variety Currency. The two wheats GS50A and AUS4930 had fewest nematodes among the wheats.
Soil type had two significant interactions, the first with variety (Fig 5.8) which
(depending upon the soil type) appeared to produce variety specific results. For instance, the number of P. thornei in Urrbrae loam with Galleon is significantly higher than with sand, whereas the number of P. thornei is greater in sand than Urrbrae loam for the triticale Currency. Also, Figure 5.9 shows how nematode inoculum density was affected differently by soil type. The number of. P. thornei was greater in Urrbrae loam for the two higher inoculum densities (200 and 400 P. thornei per plant); but for the lowest inoculum density of 100 P. thornei the number per plant was greater in sand. 74 Chapter 5 Multiolication of P. thornei
5.3.4 Discussion
From the Analysis of Variance (Table 5.8) it can be seen that few of the factors tested were significant. At harvest 1, after just one month of growth, few differences were obtained between varieties examined probably because insufficient time had elapsed for nematode multiplication. The results reflected the numbers of nematodes which penetrated from the inoculum, which was small at 5.0 - 6.57o. It was surprising that soil type at harvest I did not affect the percentage penetration significantly as a sandy soil should provide better conditions for movement (V/allace, 1963). It may be that the
Urrbrae loam had very good structure and crumb formation for movement or the containers may have been small enough to limit the contribution that movement might make to numbers penetrating.
After 2 months, the nematodes had multiplied at all initial P. thornei densities so that differences between levels of resistance had begun to show. The fact that no differences were apparent after 1 month shows that the minimum time to detect resistance is probably close to 2 months. In considering tube size, evidence from Section 5.1 comparing multiplication of nematodes on the same host species, with the exception of
Galleon, suggested smaller tubes produced greater numbers of nematodes when the same time frame was considered (Fig. 5.7). This probably reflects greater efficiency in penetration in the smaller tubes and suggests that container size could be most important in the resistance assay. An increase in inoculum density up to 400 P. thomei per plant did not seem to affect the multiplication rate of the nematode on the range of hosts tested. This confirms the findings of Nicol (1991) and suggests that the initial densities used do not alter the multiplication rate of the nematode. 75 Chapter 5 Multiplication of P. thornei
Finally, the best conditions for a resistance assay involved the use of small containers allowing for maximum nematode penetration, an initial inoculum density which is non- damaging (100 - 400 P. thornei per plant), and sufficient time between planting and harvesting (at least two months) to allow for nematode multiplication and distinction between host susceptibilities. 76 Chapter 5 Multiplication of P. thornei
5.4 Modification of Resistance Assay
5.4.1 Introduction
The findings in Sections 5.2 and 5.3 indicated that a two month assay in small tubes provided a reasonable method of distinguishing the multiplication rate of P. thornei on a range of different cereals and non-leguminous hosts. The assay has the potential to rank not only different varieties/cultivars but also to identify lines of low susceptibility, possibly inferring some degree of resistance to P. thornei. However, the distinction between the susceptible and resistant varieties may be further differentiated over a longer period than two months. In addition to identifying resistant plants, the assay may also allow investigations into the mode of inheritance of resistance.
In the following section, changes in plant susceptibility to P. thornei over time with selected potentially resistant and highly susceptible wheat varieties were examined in large and small tubes .
5.4.2 Materials and Methods
Three wheat cultivars (two potentially resistant and one susceptible) were tested over two harvest times (two and three months) in two sizes of container (small and large tubes). The wheat cultivars were selected from the previous susceptibility rankings in
Sections 5.1 - 5.3. These included GS50A (7. aestivum), awheat cultivar potentially resistant to P. thornei, and 4US4930. The latter (T. vulgare nigro-graecum) was designated by Watkins as "IRAQ 48" and was collected from wheat areas of Iraq in
1919 (M.Mackay, pers. comm.). It is a medium height, hard grained, early spring wheat. The third wheat cultivar chosen was the highly susceptible South Australian wheat, Machete (7. aestivum). 77 Chapter 5 Multiplication of P. thornei
The plants were set up as an RCBD with eight replicates as described in previous sections (5.1 and 5.2). All seeds were sterilised, germinated and selected as in Section
3.5. One seed of each cultivar with three even seminal roots of length 3 cm was grown in electrical conduit tubes of two sizes, small (2.7 cm wide by 12.5 cm high) and large
(3.7 cm wide by 12.5 cm high). Both large and small tubes were tested in order to detect possible limitations of container size due to root volume after 3 months. The tubes were filled with autoclaved Palmer soil (Section3.7) which had been heat-treated at 65'C for 45 minutes. Seedlings were inoculated (Section 3.1) with 400 P. thornei per plant in 1 ml aliquots with nematodes from canot cultures (Nicol, 1991).
Plants were placed in a controlled temperature growth room at 20"C, 12 hour day and night provided by fluorescent light tubes (65p Einsteins). Nematodes were extracted from plants harvested after 2 and 3 months using a 3-day mister extraction (Section
3.2.2) and counted (Section 3.3).
5.4.3 Results
Data were analysed as a RCBD with the original analysis showing heterogeneity of variance so the data was log transformed (log"(x+1)) (Table 5.9).
Table 5.9 : ANOVA of resistance assay with potentially resistant and susceptible wheat over 2 harvest times.
d.f m.s v.r. Prob
block 7 4.628
block.plot harvest 1 0.883 0.46 0.198 tube size I 0.191 0.10 0.753 vanety 2 49.22 25.81 <0.001 harvest.tube size I 0.0807 0.42 0.517 harvest.variety 2 7.952 4.17 0.019 tubesize.variety 2 8.786 4.16 0.013 harvest.tubesize.variety 2 0.565 0.30 0.745 residual 76(I 1.907 Total e4( I 78 Chapter 5 Multiplication of P. thornei
Fig. 5.10 : The effect of tube size on the number P. thornel per plant extracted from three different cultivars grown at2O"C in a controlled growth room.
6.0
5.5 small tube 5.0 I E large tube 4.5 5¡¡r=0.49 É 4.0 so- ol 3.5 EI EIol 3.0 ;l 2.5 è0 o 2.O
1.5
1.0
0.5
0.0 Machete GS5OA AUS493O
variery
Fig. 5.11 : The effect of harvest time on the number of P. thornei extracted from 3 different wheat cultivars grown at20"C in a controlled growth room.
6.0
5.5 + Machete -+ AUS4930 5.0 * GS50A
aÉ ã- 4.5 SED=0.49 'Et tst 91 4.0 ï Àl s.s JI
3.0
2.5
2.O harvest I harvest 2 79 Chapter 5 Multiolication of P. thornei
The two way interaction between tube size and variety was significant (Fig.5.10).
From the figure it is seen that AUS4930 and Machete exhibit higher numbers in smaller than larger tubes, however the opposite occurs for GS50A.
The two harvest times, of two and three months, had a significant interaction on the number of nematodes per plant for the three varieties (Fig. 5.11). The number of nematodes per root system increased for Machete and AUS4930 over time; however
GS50A showed reduction in P. thorn¿i numbers over time.
5.4.4 Discussion
There was a significant difference between wheat lines tested at both harvest times irrespective of tube size. The number of nematodes recovered changed only slightly for
AUS4930 whereas GS50A showed decreased numbers at the second harvest time. In comparison for the susceptible host, Machete, the numbers of P. thornei increased from the second to the third month. This implies that the distinction between resistance and susceptibility is increased as the time duration of the assay is extended.
The number of nematodes within the roots of the plants at both harvest times was less than in previous sections described in Chapter 5. It is possible that the inoculum from the carrot culture was less vigorous; however the ranking of the three varieties relative to each other was the same. If the assay was conducted for extended periods of time
(up to three months), the volume of the root system may confound the results, due to its limited size in the tubes, both small and large.
5.5 General Discussion
The results presented here clearly indicate that cereal and non-leguminous hosts tested differ in their susceptibility to P. thornei. The ranking of varieties with respect to their 80 Chapter 5 Multiolication of P. thornei susceptibility to this nematode species is statistically comparable regardless of whether the plants were grown over a whole season or for only two months after inoculation.
The Australian bread wheats were much more susceptible than most of the other cereals tested (barley, triticale, rye, oat and durum). The non-leguminous plants linseed and canola also offered some degree of resistance.
The relative susceptibilities of different hosts are determined primarily on the basis of nematode multiplication within the root system over a specific time period. It is important to note that the multiplication rate is highly dependent on the initial nematode density (Section 2.9). The use of a particular multiplication rate to define resistance is not a reliable method of distinguishing resistant plants. As reported by Phillips (1985), it is possible to standardise the initial density for test procedures, however the
"effective" density will vary. The findings presented in this chapter support this. These variations in the effective initial density include environmental conditions which may affect invasion and development of nematodes (Phillips, 1985). This makes establishment of the ranking a more appropriate method to assess resistance than absolute numbers.
P. thontei is generally assumed to be an endoparasitic plant nematode (Dropkin, 1989).
However, it should be noted that some evidence suggests that P. thornei may feed ectoparasitically, as well as endoparastically (Zunke, 1990). The ranking of the varieties in the present work was statistically comparable (Section 5.1), regardless of whether extraction was done from roots alone or from roots plus soil. This finding supports the view that P. thornei is mostly endoparasitic. Hence assessment of resistance using root systems alone seems a valid approach to the assay. 8l Chapter 5 Multiolication of P. thornei
The range of susceptibility of host plants to the closely related species P. neglectus and
P. thornei differed significantly, although wheats were generally more susceptible to both nematodes than oats and barley. These specific variety/cultivar differences have important implications for possible control strategies, given that mixes of both nematode species are frequently found in the cereal growing regions (Chapter 4).
The screening of a range of wheat lines identified two potential sources of resistance in wheat. The resistance in GS50A verifies the work of Thompson (private coÍrm. 1993), while the land race variety designated AUS4930 appears to confer some degree of resistance to P. thorn¿i in addition to the single gene for resistance to the cereal cyst nematode, H. avenae. The assay was found to give best results in small tubes over a two month time period post inoculation in a sandy soil medium using an initial P. thornei density which is non-damaging (i.e. up to 400 nematodes per plant). 82 Chapter 6 Population Dynamics qnd Yield Relations in the Laboratory
Chapter 6 Population Dynamics and Yield Relations of P. thornei in the Laboratory
6.0 General Introduction
Little is known about P. thornei population dynamics on cereals (Section 2.8). Jones
(1956) noted that the principles underlying the populations dynamics of plant parasitic nematodes can be studied in pots of various size, small field plots or microplots.
Population dynamics can be defined as the changes in the number, age, class, sex ratio and behaviour of a population through time and space, determined by inherent characteristics of the individuals, and mediated by the environmental conditions of food resources and interacting biotic agents (Ferris and \ù/ilson, 1987). An understanding of population dynamics is considered essential for the development of control measures.
Ideally, field studies should be conducted to obtain accurate information about population dynamics. However, in order to determine the effect of P. thornei alone with wheat in relation to populations dynamics of the nematode and the associated damage relations of the host, several aseptic laboratory experiments were conducted on wheat.
6.1 Population and Pathogenicity of P. thorn¿i on Machete at 25oC
6.lJ Introduction
There is no information available on the multiplication of P. thorn¿l on local wheat cultivars in South Australia. Pattison (1993) has examined changes in P. thornei multiplication on various cereal cultivars in northern NSW in different soil types and conditions. Clearly, there is a need to examine multiplication rates and damaging densities on local cultivars. 83 Chapter 6 Population Dynamics and Yield Relations in the Laboratorv
6.I.2 Materials and Methods
An attempt was made to examine the effect of different initial P. thornei densities on nematode population dynamics and the yielding capacity of the wheat cultivar
Machete. Nine nematode densities were investigated each with eight replicates grown for a period of four months.
Three hundred seeds of the wheat cultivar Machete were selected on the basis of size uniformity and were sterilised and germinated as in Section 3.5. Soil (Urrbrae Loam) taken from the Waite Institute was autoclaved at 120"C for I hour and after cooling was thoroughly mixed. It was then placed into 72 perforated (4 holes) black polyethylene bags (1l.5cm wide by 16cm high) over a layer (2-3cm) of wood chips to encourage drainage. Germinated seedlings of Machete were selected for uniformity
(three roots each about 3cm long) and one seedling was planted in each bag.
P. thornei was extracted from c¿urot cultures as described in Section 3.1 and inocula of
0, 500, 1500, 2000,3000,4000, 6000, 10000, and 15000 nematodes were used per bag with 8 replicates per treatment. The nematodes were added to the bags as close to the seedling as possible. SDW was used for the 0 nematode treatment. Plants were arranged as a CRD in a controlled growth room at 25"C with 12 hours light, intensity
300-400 pEinsteins supplied by high pressure sodium lamps. Plants were sprayed routinely for powdery mildew with Bayleton (lmlil.).
Plants were harvested 4 months after inoculation. The soil was gently washed from the root system. Nematodes were extracted over a period of 7 days using the mister extraction (Section 3.2.2) and were counted (Section 3.3). Dry weight of shoots and roots were recorded after drying at 80"C for 7 days. 84 Chapter 6 Pooulation Dynamics and Yield Relations in the Laboraton,
6.1.3 Results
Plants were very healthy till after heading which occurred at approximately 2 months after sowing. Unfortunately, due to unknown reasons many of the heads were found to be sterile at harvest. As a consequence only shoot dry weight only was recorded.
The data, number of P. thorneil plant; number of P. thorneil gplant; multiplication rate
(number P. thornei per plant/ initial P. thornei density); dry weight roots (g); dry weight shoots (g); total dry weight (g)); were analysed as a CRD. Initial density had a highly significant effect on the final number of P. thorneilplant (Table 6.1), the numbers in the plant increasing in a linear fashion with increasing density (Fig. 6.1).
Table 6.1 : ANOVA for variables measured on Machete after 4 months d.f. m.s. v.r Prob.
Variable : No. P. thorneilplant Density 8 1.483E+10 19.49 <0.001 Linear I l.l6lE+l l 152.67 <0.001 Quadratic I 1.4598+08 0.19 0.663 Deviation 6 3.917E+08 0.5r o.794 Residual s3(r 0) 7.606E+08
Va¡iable : No. P. thorneilg plant Density 8 2.5618+09 9.80 <0.001 Linear I 1.7998+10 68.86 <0.001 Quadratic I 7.363E+08 2.82 0.099 Deviation 6 29378+O8 t.t2 0.361 Residual s3( 1 0)
Va¡iable : Multiplication rate P. thornei Density 7 73.96 1.96 0.082 Residual 46(10) 37.82
Variable : Total dry weight (g) Density 8 89.49 1.04 0.42r Residual 53(10) 86.34
Va¡iable : Dry weight shoots (g) Density 8 23.389 248 0.023 Residual 53(10) 9.412
Variable : Dry weight roots (g) Density 8 56.74 1.00 0.446 Residual 53(10) 56.68
Total 6l(10) 85 Chøpter 6 Population Dynamics and Yield Relations in the Laboraton,
Fig. 6.1: The effect of initial density of P. thornei on the number of nematodes extracted per plant from Machete after 4 months.
200000 l= -4495.2+8.6288x 175000 SED = 13790 150000 d 125000 ol EI ol 100000 .cl ;l 75000 .i z s0000
25000
0 0 4000 8000 1 2000 1 6000 20000 Initial density P. thornei / plant
ßigt !.2: The_effect of initial density of P. thorn¿i on the nematode multiplication rate on Machete after 4 months (note : non-significant).
12
F y=4.2372+3.9083e-4x È 10 SED = 3.08 EI H ol 8 -Él
Èl 6 o (€ 4 (_) È = 2 ¿. 0 0 4000 8000 12000 16000 20000 Initial density P. thorneilplant 86 Chapter 6 Population Dynamics and Yield Relations in the lnboraton,
Fig. 6.3 :_The effect of initial density of P. thont¿i on the number of nematodes per gram dry Machete root.
60000 y = -2497.1+3.3826x o SED 3931 50000 = ¡r èo 40000 õ)l EI ol - ;llr c) 20000 ,Õ z 10000 0 0 4000 8000 12000 1ó000 20000 Initial density P. thornei lplant F!S. 6ra : The effect of initial density of P. thorn¿i on the dry weight of Machete shoors after 4 months. 14 SED = 1.5 bo 12 a 10 rh o 8 .tl 6 öo 0.) È 4 l< 2 0 ÊE*ÊããËã Initial density P. thornei / plant 87 Chapter 6 Population Dynamics and Yield Relations in the Laboraton, When nematode multiplication rate was plotted against the initial density we see a gradual increase in multiplication rate as initial density increased (Fig. 6.2). Although this was non-significant (P<0.05) the Fig. 6.2 is still included. The number of P. thornei per gram of plant had a highly significant linear relationship with initial nematode density (Fig. 6.3). Significant differences in dry weight of shoots were obtained (Table 6.1) but from Fig. 6.4 it can be seen that dry weight increased with increasing density up to 6000 P. thontei per plant but was lower for higher initial densities. Neither total dry weight nor dry weight of roots were affected significantly by increasing initial density (Table 6.1), 6.I.4 Discussion Unusual effects and relationships were demonstrated in this experiment. Neither multiplication rates of the nematodes, nor the response of growth to increasing initial density of nematodes gave responses that would be normally expected. An explanation for both these unexpected results could be that initial penetration of the roots was extremely poor and although large numbers of inoculum were added only a very small percentage actually penetrated the roots. This would mean that numbers in roots were very small and that the very low initial density meant that stimulation of growth occurred. Multiplication rates normally drop rapidly as initial density increases (Brown and Kerry, 1987). Similarly, growth of plants is normally increasingly inhibited as initial density increases (Nicol, 1991). Nusbaum and Ferris (1973) suggest conditions unfavourable to the host crop can indirectly reduce the maximum rate of multiplication and the equilibrium density of the nematode compared to those found under favourable conditions. At low levels nematode infestations are known to produce a stimulus in growth (Chitwood and Buhrer, 1946; Chitwood and Feldmesser, 1948; Chitwood and Esser, 1957 and Peter, 196I; in Wallace, 1963). Tillering was increased in oats with 88 Clupter 6 Population Dynamics and Yield Relations in the Laboratom Ditylenchus dipsaci (Dunning, 1954 in Wallace) and in tall fescue with Paratylenchus projectus (Coursen and Jenkins, 1958; in W'allace, 1963). Species of the sedentary nematode, Heterodera are noted for causing increases in root growth and low initial densities (W'allace, 1963). 89 Chapter 6 Population Dynamics and Yield Relations in the Laboraton, 6.2 Soil Type Relations wWlectus 6.2.1 Introduction Given the results in the previous section, it is important to examine the penetration efficiency of the nematode. P. thomei and P. neglectus usually inhabit different soil types; P. neglectøs is found more commonly in lighter soils while P. thornei more commonly inhabits heavier, clay based soils (Section2.6.2). However, the optimal soil conditions for movement (Wallace, 1963) suggest both nematodes should move well in sandy soil. This section has two components. The first is an experiment using P. neglectus instead of P. thornel investigating nematode penetration in relation to soil type and container size. P. neglectus was used because sufficient numbers of P. thornei were not available. The second experiment examines penetration of both P. thornei and P. neglectus in relation to soil type and the efficiency of the extraction process. This latter work was done in collaboration with Mr. Abdolhossein Taheri, Department of Plant Science, Waite Campus. 6.2.2 Materials and Methods Machete wheat seeds were sterilised, germinated and selected as in Section 3.5. For the first experiment 2 soil types; Urrbrae Loam (Ul) a red brown earth from the 'Waite Institute with a high clay content, and Palmer sand (Ps) a very sandy soil from the farming region of Palmer were used (Section 3.7). The Ul was heat treated at 100oC for t hour and the Ps at 65"C for 45 minutes. Both soil types were mixed separately and all the Ps and half the Ul were sieved through aZmm sieve. For the first experiment two containers were used, large perforated (4 holes) black polyethylene bags (11.5cm wide by 16cm high), and electrical conduit tubes (3.7cm wide by I2.5cm high). Equal numbers of each type of container were filled with each soil type (Ul non-sieved, Ul sieved, Ps sieved) and 1 seedling was planted in each 90 Chapter 6 Population Dynamics and Yield Relations in the Laboraton, container as described previously (Section 3.5). The seedlings in the tubes were inoculated with 170 P. neglectusl ml of water and the plastic bags with 1500 P. neglectuslml, 1 week after sowing. Plants were arranged as a CRD, with the bags and tubes separated due to space limitations, but soil type was randomised within the container sizes. Ten replicates of each soil type and container size were grown in a controlled temperature room at 20"C with 12 hour day length and light intensity of 65 pEinsteins. The tubes were supported by a wire grid and the bases were embedded into potting soil while the bags were placed on a metal tray. Plants were harvested 1 and 3 weeks after inoculation. Nematodes were extracted in a mister (Section 3.2.2) for 3 days and counted (Section 3.3). In the second experiment four different soil types were used, two with a high clay content and two with a sandy composition. These were Urrbrae loam non-sieved (Ulns), Urrbrae Loam sieved (Uls), Palmer sand sieved (Ps) and Roseworthy sand (Rs) also sieved through a 2mm sieve. The four soil types were tested for penetration efficiency of the 2 nematodes, P. thornei and P. neglectus. Plants were similarly inoculated, and the inoculum density was 2000 P. thornei or P. neglectus per plant. Plants were set up as a Split Plot with 6 replicates for each nematode species, soil type and harvest time. The plots contained either P. thornei or P. neglectus,whTle within each subplot the soil type was randomised. The plants were grown in 300m1 plastic cups (Plate 6.1) without drainage holes and were harvested 1 week after inoculation. The nematodes were extracted in a mister (Section 3.2.2) for a period of 4 days and counted (Section 3.3). The nematodes remaining in the root system were also counted after staining with acid fuschin (Section 3.4.1). 9l Chapter 6 Population Dynamics and Yield Relations in the Laboraton, 6.2.3 Results The data from both experiments was analysed by ANOVA. For the first experiment, because the tubes and pots were inoculated with different nematode densities, the data was converted to percent penetration for the analysis, presented in Table 6.2. Table 6.2 : ANOVA from the first experiment: effect of soil type and volume on the penetration efficiency of P. neglectus. m.s. v.f . kob. contarner I 0.1 8 129 6.92 0.010 soil 2 0.48985 18.70 <.001 harvest time I 0.1048 l 4.0 0.048 container.soil 2 0.10545 4.02 0.021 container.harvest time 1 0.02117 0.81 0.371 soil.harvest time 2 0.02702 1.03 0.361 container.soil.harvest time 2 0.00720 0.27 0.760 Residual e3( 15) Total 104(15) There was a significant interaction effect between container and soil type. Also harvest time significantly affected penetration. The significant interaction between container and soil type arose because container type had no influence with Ps but with the Ul (sieved or non-sieved) the percentage penetration was significantly higher in tubes than in plastic bags (Fig. 6.5). There were significantly more nematodes in the roots at the 3 week harvest (Fig. 6.6), but no significant interaction between harvest time and container size or harvest time and soil type. The ANOVA for the second experiment, analysed as a SPD, is presented in Table 6.3. The three variables analysed were total number nematodes per plant (mister extraction plus stained nematodes in roots), mister extracted nematodes only per plant, and stained nematodes in roots only per plant. All results were analysed as percentage penetration. The ANOVA of the total nematodes showed that P. neglectøs and P. thornei acted similarly regardless of soil type. Overall, the actual effectiveness of the original inoculum was low. Fig. 6.7 illustrates the significant interaction between soil type and 92 Chapter 6 Population Dynamics and Yield Relations in the Laboraton, the total nematodes extracted from the soil. Sandy soil was by far the best medium to allow maximum penetration within the roots (up to OVo). However, the Ul was very inefficient, particularly if unsieved, with fewer nematodes (only 5Vo) penetrating the roots. Table 6.3 : ANOVA from the second experiment: effect of soil type and extraction technique on penetration efficiency of P. thontei and P. neglectus. (data analysed as a percentage of the initial inocula) m.s, v.r Prob. Variable : Total Nematodes per plant block stratum 5 0.01568 block stratum nemtype 1 0.00004 0.00 0.956 Residual 5 0.01283 block.wplot.subplot stratum soil 0.37377 2r.33 <0.001 nematype.soil 3 0.00916 0.52 0.670 Residual 2e(t) 0.0t752 Total 46(l) Variable : Mister nematode extraction only per plant block stratum 5 0.002662 block.wolot stratum nemtype I 0.035100 t2,06 0.018 Residual 5 0.00291I block.wplot.subplot stratum soil 3 0.026381 8.84 <0.001 nematype.soil 3 0.007924 2.64 0.067 Residual 29(l) 0.002985 Total 46(l) Variable : Stained nematodes in roots only per plant block stratum 5 0.006524 block.wolot stratum nemtype I 0.037630 6.75 0.048 Residual 5 0.005573 block.wolot.subolot stratum soil 3 0.021152 24.20 <0.001 nematype.soil 3 0.023475 2.69 0.065 Residual 29(l) 0.008742 Total 46/L\ 93 Chapter 6 Population Dynamics andYield Relations in the Laboratom Fig. 6.5 : The effect of soil type and container size on the penetration of P. neglectus in Machete roots. e35É ë¡o I tubes o E bags Ë25 Lr SED .051 o = E,o 9t Hl ls l.;)l àtr 91Ìt 10 O.l ba5 0 a) 0) c) (.) 0.) c) û Ø Ø I o d o Ø () q)L d (ú Ð (ü c) CÚ .o ts Fig. 6.6 : The effect of harvest time on the penetration of P. neglectus in Machete roots. 25 SED = .03 Ë(! ë20(^t EI OI (L)l Ë15ot 1t 0.1 810 crt L ÊÃo) a(l)v ñ 0 0 I week 3 week Time after inoculation 94 Chapter 6 Population Dynamics and Yield Relations in the Laborato4, Fig. 6.7 : The effect of soil type and nematode extraction technique on the numbers of Pratylenchøs in Machete roots. 40 ) I mister extraction ) SED=5.4 C) 35 o E Pratvlenchus remaining in root 30 SED=3.8 N total number nematodes 25 SED=2.23 Io o 20 63 o) L 15 €o) l0 (ü o 5 xA 5\ 0 Urrbrae loam Urrbrae loam Palmer Roseworthy (non-sieved) (sieved) sand sand Fig 6.8 : The effect of mister extraction and staining nematodes in the remaining Machete roots on the proportion of P. neglectur and P. thornei. ) 20.0 o 17.5 I mister extraction SED=2.16 d 15.0 remalnlng ln root SED=1.56 12.5 o) 10.0 (É o L 7.5 o o 5.0 c'd C) 2.5 òa 0.0 Pratylenchusthornei Pratylenchusneglectus Nematode species The ANOVA of the total nematodes showed that P. neglectøs and P. thornei acted similarly regardless of soil type. Overall, the actual effectiveness of the original Chapter 6 Population Dynamics and Yield Relations in the Laboraton, Plate 6.1 : The experimental setup for the second experiment used to assess the penetration of P. thornei and P. neglect¿rs over a range of soil types after 3 weeks. 300m1 plastic cups were ananged as a SPD and grown in a controlled growth room at 2O"C. Light was supplied by flourescent light tubes (65p Einsteins) and plants watered with tap water whenever necessary. Plate 6.2 : A representative stained Machete wheat root system, 2L days post inoculation, initially inoculated with 2000 P. thornei in Urrbrae loam (non-sieved). Evidence of masses of P. thornei adults (represented by the dark pink area) and nematode eggs (oval shaped rods) in cortical cells of seminal root system. (lcm = 50pm) 95 Chapter 6 Population Dynamics qnd Yield Relations in the Laboraton, inoculum was low. Fig.6.7 illustrates the significant interaction between soil type and the total nematodes extracted from the soil. Sandy soil was by far the best medium to allow maximum penetration within the roots (up to 40Vo). However, the Ul was very inefficient, particularly if unsieved, with fewer nematodes (only 57o) penetrating the roots. Further analysis (Table 6.3) of the two components of the total number of nematodes per plant (mister extracted and stained nematodes within the root) revealed there was a significant species effect, both with the mister extraction and the numbers of stained nematodes remaining in the root system (Fig. 6.8). From the mister extraction 42Vo of P. neglectus left the root system in 4 days, but the number of P. thornei leaving the root was significantly less, almost 3 times fewer (l6Vo). However, there were significantly more P. thornei left inside the roots (84Vo) (Plate 6.2), compared with P. neglectus (58Vo). 6.2.4 Discussion Infection depends on the movement of nematodes through soil and their possible attraction to roots. As the same cultivar of wheat was used for both experiments, the attractants did not vary, but the movement through soil did. The percentage penetration in both experiments was higher in sand (Palmer and Roseworthy) than in sieved Urrbrae loam than in unsieved Urrbrae loam, so that particle size influenced movement. Why sieving improved penetration is not clear, but it may have changed particle or crumb size or removed possible toxic materials in the organic matter. The first experiment showed that larger containers, such as the bags, interacted with soil type to influence percentage penetration. If the nematode had difficulty in moving in the soil type then larger volumes of soil exaggerated this difficulty. The differences in percentage penetration between bags of Ul in bags and tubes was much greater than for the Ps. Thus container size can influence percentage penetration in unfavourable 96 Chapter 6 Dynamics and Yield Relations in the Laboratom soils. Nevertheless, the best penetration was in sand and so particle size was more important than container size though both may influence penetration. The best penetration would probably be obtained in small tubes in sand. In addition, the first experiment showed that the increase in numbers in roots at the three week harvest could have been due to further penetration or to multiplication of the nematodes in the roots. At20"C, there was probably time for any adult females which penetrated to lay eggs and for them to hatch. There is no way to distinguish between these possibilities. The second experiment reconfirms the finding that sandy soil provided the best medium for maximum penetration of either nematode species, P. thornei or P. neglectus. However, although the data presented in this Section reveal the total number of nematodes per plant (mister plus remaining in roots) do not vary between species, the individual components vary significantly. The implications of these results need to be considered. The previous experiment (Section 5.2) assessing the multiplication of P. thornei and P. neglectus over a range of hosts and comparing and ranking respective cultivars was done principally on the basis of the number of nematodes extracted per plant. As a result the comparison of numbers is not strictly comparable. Unfortunately to stain and count all the nematodes inside root systems would be too time consuming. P. neglectus left the roots much faster than P. thornei, this may possibly be associated with differences in species mobility. It is important to understand the nematode penetration in different soil types and the extraction efficiency of the method used. If comparative data are required a standardised method is needed. 97 Chapter 6 Population Dynamics and Yield Relations in the Laboraton, 6.3 Population and Pathogenicity of P. thornei on Machete at 2O"C 6.3.1 Introduction An initial attempt (Section 6.1) was made to examine the effect of P. thornei density on population dynamics and yielding capacity of the wheat cultivar Machete. Results suggested the initial densities used in the soil medium tested were too low to limit multiplication of the nematode and cause damage to the host. The previous section established unsieved Urrbrae loam as a poor medium for penetration and movement by both P. thornei and P. neglectus, and that reduction in container size could overcome part of these problems. However, the use of small containers may limit growth of the plant at later stages of development and this could cause problems in understanding the relationship between the nematode and the plant. Given that soils like Unbrae loam tend to be associated with the distribution of P. thornei in the field, it may be possible to use increased initial densities with this soil to overcome some of the problems associated with penetration. These aspects are examined in this section. 6.3,2 Material and Methods Machete seeds were sterilised, germinated and selected as in Section 3.5. Urrbrae loam was autoclaved at 120"C for t hour, allowed to cool, mixed thoroughly, sieved through a2mm sieve and placed in electrical conduit tubes (2.7cm wide by 12.5 cm high) (Section 5.1). One week after sowing 9 replicates of individual seedlings were inoculated with P. thornei densities of 0,5000, 10000, 15000,20000,25000 and 30000 in lml of water. Plants were grown at ZO"C with 12 hours light at an intensity of 65 pEinsteins and harvested 3 and 9 weeks after inoculation. Thus the design was a CRD where the seven densities of the nematode by the two times of harvest was relplicated nine times to give a total of 126 individual plants in tubes. Dry weights of shoot, root and total plants were determined by placing individual plants in alfoil trays in a drying 98 Chapter 6 Pooulation Dynamics and Yield Relations in the Laboraton, oven at 80"C for 7 days. Nematodes were extracted in a mister (Section 3.2.2) over 3 days and counted (Section 3.3). 6.3.3 Results The ANOVA of all variables is presented in Table 6.4. There was a significant 2-way interaction between initial density and harvest for the number of nematodes per root system and the multiplication rate (final no. P. thornei per planl initial density of P. thornei infected per plant), which can be seen in Figs. 6.9 and 6.10. At harvest 1, there was little change in the numbers of nematodes per plant until higher initial densities were reached, while at harvest 2 the plants inoculated with the highest density had significantly higher numbers. A cubic relationship was found to best fit the data at both harvest times (P<0.05). The multiplication rate of P. thornei for harvest I showed little change with initial density, but by harvest 2, there were considerable differences. The number of nematodes per gram root was found to increase over initial density, irrespective of harvest time, although the magnitude of this increase was reduced as density increased (Fig. 6.11). A significantly higher number of nematodes was present within wheat roots at harvest 2than harvest I (Fig. 6.12). There was no significant 2-way interaction between initial P. thornei density and harvest for the total dry weight of the plant. Furthermore, density was not found to effect the total dry weight, however there was a significantly higher total dry weight at harvest 2than harvest 1 (Fig. 6.13). A break down of total dry weight of plants to weight of roots and shoots revealed a highly significant 2 way interaction of P. thornei initial density by harvest for both variables (Figs. 6.14, 6.L5). Shoot growth was significantly higher at harvest 2 than harvest 1 for all densities, however at higher initial densities for both harvests there was a declining trend. At high initial nematode densities beyond 20,000 per plant, the roots weighed significantly less at harvest 2than at harvest 1. At harvest 1 root growth 99 Chapter 6 Pooulation Dynamics and Yield in the Laboratorv increased with densities up to 20000 P. thornei per plant, but thereafter was not seen to alter. Harvest 2 showed very little change in root growth over density. There was no evidence of damage to the root system by the nematode at both harvest times. Table 6.4 : ANOVA for variables measured on Machete after 3 and 9 weeks. d.f. m.s. v.r Prob. Va¡iable :No. P. thornei.þlant density 6 43198+07 t8.23 <0.001 ha¡vest I 2.3398+08 98.76 <0.001 density . harvest 6 6.7728+06 2.86 0.013 Residual 108(4) 2.369E+08 Total tzt(4) Va¡iable:No. P. thomeilg plant density 6 5.3808+10 4.55 <0.001 harvest I 3.3778+Il 28.56 <0.001 density . harvest 6 1.834E+10 1.55 0.169 Residual r08(4) 1.182E+10 Total tzr(4) Variable :Multiplication rate of P. thorneilplant density 5 0.17895 12.78 <0.001 harvest I t.94576 r38.94 <0.001 density . harvest 6 0.21555 15.39 <0.001 Residual e2(4) 0.01400 Total 103(4) Va¡iable : Total dry weight/plant (g) density 6 0,000s89 0.45 0.846 harvest 1 1.053978 800.06 <0.001 density . harvest 6 0.000843 0.64 0.698 Residual 10s(7) 0.001317 Total I 18(7) Va¡iable : Dry weight shoots (g) density 6 0.004051 4.t6 <0.001 harvest I 1.07025r 1098.26 <0.001 density . harvest 6 0.002861 2.94 0.01l Residual tto(2) 0.000974 Total r23(2) Variable : Dry weight roots (g) density 6 0.002486 16.26 <0.001 harvest I 0.000131 0.86 0.356 density . harvest 6 0.001643 10.75 <0.001 Residual r0s(7) 0.000152 Total I I 8t7) 100 Chapter 6 Population Dynamics and Yield Relqtions in the lnboraton, Fig. 6.9 : The effect of initial density of P. thornei and harvest time on the number of P. thornei extracted from Machete. 8000 7000 -ù harvestl + harvestz 6000 SED=726 5000 ê. clol LI 4000 €lol dl : 3000 z 2000 1000 0 0 10000 20000 30000 Initial density P. thornei/plant Fig. 6.10 : The effect of initial density P. thornei and harvest time on nematode multiplication rate on Machete. 0.8 -- hNest I 07 * ha¡vest2 SED=0.06 0.6 sol !tél ôl €l 0.5 Èr o é 0.4 03 o6 q 0.2 ¿ 0.1 0_0 5000 l0m0 15000 20000 25000 30000 Initial density of P.thornei/plant and Yield Relations iill[::L4:.:, Fig. 6.11 : The effect of initial density of. P. thorn¿i on the number P. thornei per gram dry weight Machete. 200000 sEÞ51260 175000 150000 Ë I 125000 Þ¡) 'gr bt 100000 ql Èl 75000 zci 50000 25000 0 0 5000 10000 15000 20000 25000 30000 35000 Initial P. thornei density/plant Fig. 6.12 : The effect of harvest time on the number of P. thornei per gram dry weight Machete. 160000 SED=19,374 140000 : o 120000 ào ol Él ät 100000 EI ;l zc; 80000 60000 40000 harvest I harvest 2 r02 Chapter 6 Population Dynamics and Yield Relations in the Laboratory, Fig. 6.13 : The effect of harvest time on the total dry weight of Machete. 0.30 sED{.0065 0.25 èo E cJ o. 0.20 èo ;o 015 d Fo 0.10 0.05 harvest I harvest 2 Fig. 6.14 : The effect of initial P. thornei density and harvest time on the dry weight shoots per Machete plant. 0.30 4 hNestl 0.25 * hæest2 à0 sED{.015 0.20 -ê. I o ô 0.15 Þ0 o ¡ 0.10 à â 005 0.00 0 5000 10000 1s000 20000 25000 30000 Initial density P. thomei/plant 103 Chapter 6 Pooulation Dynamics and Yield Relqtions in the Laboro.ton, Fig. 6.15 : The effect of initial P. thornei density and harvest time on the dry weight roots per Machete plant. 0.06 -+ harvestl + harvest2 bo 005 SED=O.0058 ect) 0.04 o o ¡i bo 0.03 o È >\ o.o2 0.01 0 10000 20000 30000 Initial density P. thornei/plant 6.3.4 Discussion Once again it appears that P. thornei did not cause yield damage to Machete with regard to the growth parameters measured under the experimental conditions used here. Although from previous experiments sieved Urrbrae loam gave a penetration efficiency of 22Vo with P. neglectus in polyethylene tubes, it appeared to range from 8-l2%o over the densities used in this experiment. This is very low and may possibly explain why losses in yield are not found. The fact that little damage on the roots was evident also supports this. In contrast to reductions in yield, stimulatory responses in shoot growth occurred as initial density increased. This stimulus in shoot growth confirms previous findings in Section 6.1. Stimulation of root growth appeared at harvest 1, but by the second harvest at 9 weeks this stimulus had ceased. This is in contrast to the work of Nicol (1991), where the wheat host Warigal suffered significant yield reductions with 15,000 P. thornei per plant by 9 weeks, also using Urrbrae loam grown in similar conditions. 104 Chapter 6 Population Dynamics and Yield Relations in the l-aboraton, P. thornei displays attributes of the models proposed for nematode population dynamics (Section 2.9). P. thornei populations in the plant increased with increasing initial P. thornei density at both harvest times. However, initial densities failed to reach the equilibrium level where final and initial nematode populations are equal (Fig. 2.3). The density dependent relation for plant parasitic nematodes with regard to multiplication rate (Brown and Kerry, 1987) holds true for p. thontei (Fig. 6.10). At harvest 1, there was probably little time for multiplication of P. thornei. However, by harvest 2 (9 weeks) the low initial densities led to the maximum multiplication rate, and as initial density increased the multiplication rate of P. thornei was significantly reduced, presumably due to increased competition between individuals accompanied with a decreasing food resource. At 3 weeks initial densities of 20,000 P. thornei per plant caused a stimulus in growth. This is possibly a response to initial penetration. Between the two harvest times the nematodes multiplied, reduced stimulation of growth of the plant and in turn reduced nematode multiplication. In this instance, no difference was found between the growth of inoculated and control plants, but if this experiment had been conducted for a longer period this may have been changed. 105 Chapter 6 Population Dynamics and Yield Relations in the lnboratory 6.4 Population Dynamics and Pathogenicity of P. thornei on Warigal at20"C 6.4.1 Introduction This experiment examined the effect of initial P. thornei density on the nematode population dynamics and the yielding capacity of wheat for the whole growing season. The previous two experiments were using the wheat cultivar Machete (Section 6.1, 6.2). Results presented in Section 6.1 suggested that the initial density did not limit the P. thornei multiplication rate after 4 months in polyethylene bags. Further investigations (Section 6.3) revealed that poor nematode penetration could have accounted for this. Adjusting the experimental technique in order to improve penetration efficiency, accompanied with a range of nematode densities used, indicated that the multiplication rate could be limited with 10000 P. thornei per plant after 9 weeks of growth in sieved Urrbrae loam. However, reduction in growth of the host, Machete was negligible in the presence of P. thornei . In contrast, Nicol (1991) 'Warigal, demonstrated yield reductions on a similar wheat host, over the same time frame and with similar soil. This experiment examined the population dynamics and yield relations of the wheat cultivar Warigal 4 months post inoculation with P. thornei. 6.4.2 Materials and Methods The wheat cultivar Warigal was selected due to its known intolerance to P. thornei (Nicol, l99l; Taylor, pers. comm.). Warigal seeds were sterilised, germinated and selected as in Section 3.5. Sand collected from Palmer was heat treated at 65"C for 45 minutes and allowed to cool. It was mixed thoroughly and sieved through a 2mm sieve. The sand was then placed into eighty l4cm diameter pots with wood chips in the base (2-3 cm height) to enable drainage. Selected Warigal seedlings were placed centrally in the pots (1 seedling/poÐ. P. thornei was extracted from carrot cultures as described in Section 3.1. One week after sowing 8 replicates of individual seedlings were inoculated with IO P. thornel densities (0, 500, 1000, 3000, 5000, 7000, 9000, 12000, 15000 and 20000) in lml of water. Plants were ¿uranged as a CRD and grown r06 Chapter 6 Population Dynamics and Yield Relqtions in the Laborato4, atzO"C with 12 hours light at 65p Einstein's intensity (Plate 6.4). They were harvested 4 months after inoculation. The following parameters of growth were recorded for individual plants: number of leaves on the main tiller of each plant, number of tillers per plant (excluding main tiller); maximum height of shoots per plant; number of seminal roots per plant; maximum length of seminal roots per plant; number of nodal roots per root system; number of seeds per main tiller head; dry weight heads per plant; dry weight shoots per plant (g); total foliar dry weight (g) (dry weight fertile heads per plant + dry weight shoots per plant); dry weight roots per plant (g); total dry weight per plant (g) (total foliar dry weight + dry weight roots per plant); days to anthesis; number of nematodes per root system; number of nematodes per gram of root; nematode multiplication rate (number of nematodes per root system/ nematode initial inoculum density). Dry weight of leaves and roots were determined by placing individual plants in alfoil trays in a drying oven at 80"C for 7 days. Nematodes were extracted in a mister (Section 3.2.2) over 3 days and counted (Section 3.3). Plants were also checked for any evidence of lesioning on the root systems. Nicol (1991), similarly conducted an experiment examining the pathogenicity of P. thontei on Warigal in small electrical conduit tubes (2.7cm width x 12.5cm height) using 2 nematode densities 1000 and 15000 P. thonnei per plant harvesting plants at 5 and 9 weeks. Eleven comparable quantitative characters were examined in this experiment. 6.4.3 Results All results were analysed as a CRD, however because of the number of variables, only the F probabilities from the ANOVA are given (Table 6.5). Simple linear and polynomial regressions were fitted to the data where the initial P. thornei density was lol Chapter 6 Population þnamics and Yield Relations in the Laborato1, found to significantly affect the variable. In addition Table 6.5 gives some reference to the comparable variables examined by Nicol (1991). The initial density of P. thorneihad no significant effect on the number of tillers per plant (excluding main tiller), maximum height (cm) of shoots per plant, number of seminal roots per plant, maximum length of seminal roots per plant, dry weight heads per plant (g) and total foliar dry weight (g). Density of P. thornei also had no significant effect on the anthesis date of Warigal. Table 6.5 : Summary ANOVA of the significance of the growth variables measured (in addition to comparable parameters previously examined by Nicol, 1991). Fig. Nlcot r99l llumber leaves on the main tiller of each plant <0.001 linear 6.16 Y Number tillers oer olant lexcludins main tiller) 0.064 Y Maximum height shoots per plant(cm) 0.295 Y Number seminal roots oer olant 0.085 Y Maximum lensth seminal roots per plant(cm) 0.783 Y \umber nodal roots Der root system 0.002 cubic 6.17 Y \umber srains oer main tiller head <0.001 cubic 6.18 N \umber heads per plant 0.056 linear 6.19 N Drv weisht heads oer olantls) 0.1 34 N Dry weight shoots per plant(s) 0.059 linear 6.20 Y Iotal foliar dry weight(e) 0.099 N Drv weisht roots Der plant(e) 0.029 ouartic/linear 6.2t Y Total dry weisht per plant(s) 0.070 ouartrc 6.22 Y Days to anthesis o.427 N Number P. thomei per root system <0.001 cubic 6.23 Y Number P. thontei per s dry root <0.001 linear 6.24 Y P. thornei multiplication <0.001 ouartlc 6.25 N Y = previous variable examined N - previous variable not examined - = not applicable The number of leaves on the main tiller of each plant was found to decline significantly with greater than 5000 P. thornei per plant (Fig 6.16), with a 3OVo reduction in leaf number at 20000 P. thornei per plant. The number of nodal roots was significantly affected (<0.002) by P. thomei density as illustrated in Figure 6.17. This significance was due to a lTVo peak in growth stimulation at 3000 P. thornei per plant followed by a 108 Chapter 6 Population Dynamics and Yield Relations in the Laborato4, gradual decline in the number of nodal roots per plant to a lTVo reduction (from 11 nodals to 9) at the highest P. thornei density. Fig. 6.L6 : The effect of initial P. thornei density on the number of leaves on the main tiller of each Warigal plant. 9.0 ÉÉ À 8.5 SED=O.329 () 6 C) +i 8.0 lr o 7.5 7.0 cË E 6.5 (t) a) 6.0 od 5.5 zd 5.0 0 5000 1 0000 1 5000 20000 Initial density P. thorneilplant Fig. 6.17 : The effect of initial P. thornei density on the number of nodal roots per Warigal plant. l5 SED=1.3 t4 l3 d o. t2 o Eo (Ë ô 11 j z t0 9 8 0 5000 10000 15000 20000 Initial density P. thomei/plant 109 Chapter 6 Population Dynamics and Yield Relations in the Laboraton, Fig. 6.18 : The effect of initial P. thornei density on the number of seeds on the main Warigal tiller. 34 SED=2.38 832o -g 30 É 'ã 28 Ë26 .to) ci 24 z 22 20 0 5000 10000 15000 20000 Initial density P.thornei /plant Fig. 6.19 : The effect of initial P. thornei density on the number of heads on each Warigal plant. t.4 SED=O.15 r.3 CË t.2 6 c) zd 1.1 1.0 0.9 0 5000 10000 15000 20000 25000 Initial density P. thornei/plant 110 Chapter 6 Population Dvnamics and Yield Relations in the Laboraton' Fig. 6.20 : The effect of initial P. thornei density on the dry weight of shoots per Warigal plant. 1.5 SED=O.14 bo t.4 d o. ct) 1.3 o (t) 1.2 bo (¡) È 1.1 >r 1,0 0.9 0 5000 10000 15000 20000 Initial density P. thornei/plant fig. 6.21 : The effect of initial P. thornei density on the dry weight of roots per Warigal plant. SED=O.07 0.4r äo (t) (! 0.36 Þ. ct) o 0.31 t ào C) 0.26 È ! â o.2t 0.16 0 5000 10000 15000 20000 Initial density P. thorneilplant 111 Chapter 6 Population þrygmics and Yield Relations in the Laborato6, I'ig.6.22: The effect of initial P. thornei density on the total dry weight per Warigal plant. 3.3 SED=O.28 3.1 Þo cl \.o, 2.9 bo o 2.7 È 2.5 (s Fo 2.3 2.t 0 5000 10000 15000 20000 Initial density P. thornei/plant Fig, 6.23 : The effect of initial P. thornei density on the number of nematodes extracted per gram V/arigal. 100000 SED=18272 y= 1.0931e+4+3.6779x 80000 o o bo 'õr 60000 olEI -cl ;l 40000 z 20000 0 0 5000 1 0000 1 5000 20000 25000 Initial density P. thorneilplant lt2 Chapter 6 Population Dvnamics and Yield Relations in the Laboraton, Fig.6.24: The effect of initial P. thornei density on the number of P. thomei per gram V/arigal. 20000 SED=3603 o 16000 ct)à cn o 12000 ¡ro (l)l Él ol 8000 -cl ;l: z 4000 0 0 5000 10000 15000 20000 Initial density P. thornei/plant Fig. 6.25 : The effect of initial P. thornei density on the multiplication of P. thornei per V/arigal plant. 9 ê. I SED=O.95 ot Ét 7 olLl ;l-cl 6 (.) 5 cÉ ¡< 4 O J a 2 =f z I 0 0 5000 10000 15000 20000 Initial density P. thorneilplant Changes in the number of seeds per main tiller head was highly significant and the number of heads per plant was marginally significant. The number of seeds (Fig. 6.18) was significantly reduced from 28 to 21 seeds per head, a25Vo reduction by densities at 113 Chapter 6 Population Dynamics and Yield Relations in the Laborato1, and greater than 12000 P. thornei per plant. In contrast, the number of heads was increased at densities greater than 12000 P. thornei per plant (Fig. 6.19). Dry weight of the shoots per plant (g) was found to be marginally significant (Fig. 6.20). Similarly, the dry weight of the roots per plant (g) was found to be highly significantly affected by increasing P. thornei density (Fig. 6.21). Both roots and shoots display similar trends at low initial densities with a stimulus in growth up until 3000 P. thornei per plant with the stimulus greater with roots (43Vo) than shoots (I7Vo). This is also reflected in changes with total dry weight (Fig. 6.22) although this was non-significant. At P. thornei densities above 7000 per plant there was a reduction in growth between l0-I6Vo in the shoots (Plate 6.4). However, with the roots, growth was stimulated until P. thomei densities beyond 12000 per plant were reached, with plant growth reduced by 22Vo only at the highest density of 20000 P. thornei per plant (Plate 6.5). A highly significant effect on the number of P. thorneiper root system, per dry gram of plant and P. thornei multiplication is illustrated in Figs. 6.23, 6.24 and 6.25. As evident from Figs. 6.23 and 6.24, an almost linear relationship exists with the number of P. thornei extracted per root system and per gram root in relation to initial density up until the initial density of 7000 P. thornei per plant. The population dynamics of the nematode are further illustrated in Fig. 6.25, where the multiplication rate (number of P. thorn¿i extracted from the root system divided by the initial density of P. thornei) in relation to initial nematode density is shown. Very low initial densities were associated with a high nematode multiplication of 8 times, but at 1000 P. thornei per plant there was a severe restriction in multiplication to 2 times. Qualitative observation of the root system for evidence of lesioning was found to be variable between replicates. Evidence of damage was rarely seen at densities up to Chapter 6 Population Dynamics and Yield Relations in the Laboraton, Plate 6.3 : The experimental set up of the pots used to assess the relation between initial density and growth of the wheat cultivar Warigal. Plants within pots at various initial P. thornei densities were ananged as a CRD and grown in a controlled growth room at 20"C. Light was supplied by fluorescent light tubes (65p Einsteins) and plants watered with tap water whenever necessary. Plate 6.4 : Representative replicates of the wheat cultivar, Warigal over a range of initial P. thornei densities after 4 months. At low initial densities (< 5000 P. thorneilplant) a stimulus in shoot fresh weight can be seen. The higher initial densities were associated with a conesponding decrease in shoot dry weight. Thq maximum height per plant showed a trend of decreasing with increasing initial density, however this was not significant. Plate 6.5 : Representative replicates of the wheat cultivar Warigal for 0 nematodes (left), 12,000 P. thorneilplant (middle) and the highest initial density, 20,000 P. thorneilplant (right). A highly stimulatory response to growth was seen up to 12,000 P. thorneilplant but a reduction in root growth is found with the highest initial density. This reduction is highly attributable to a loss of the finer branching lateral roots. I J'- \,l \ l¡ I i I { I \ \ i l t\ ì I l; I i,' rr I /'\ \ @ @ @ @ CONTROL 12000 20000 tt4 Chapter 6 Population Dynamics and Yield Relations in the Laboratory 3000 P. thornei per plant. This damage may be absent due to the compensatory stimulation of root growth (Fig. 6.21). Densities above and beyond 5000 P. thornei per plant were associated with some dark brown necrotic lesioning, predominantly on the seminal root system. Most replicates of the high densities (above 15000 P. thornei per plant) demonstrated extensive lesioning and a lack of fine lateral root growth, as illustrated in Plate 6.5. 6.4.4 Discussion The results clearly demonstrate that P. thornei can significantly affect the growth of the host V/arigal. In addition, the population dynamics demonstrated here agree with models previously documented for other plant parasitic nematodes (Section 2.9). The results, although conducted in the laboratory with unrestricted water, suggest that P. thomei is able to multiply by a factor of 8 during the growing season at low initial densities (500 P. thomei per plant). Higher densities reduce nematode multiplication, with2.l2 times multiplication at 1000 P. thornei per plant, and only 1.1 at 12000 P. thomei per plant. Densities beyond 15000 P. thornei per plant inhibit multiplication, so that P. thornei is unable to replace its initial population. Considering the relation between initial and final populations, P. thornei was found to increase in a linear fashion reaching an equilibrium density at the initial density of 12000 P. thornei per plant. This equilibrium can be attributed to increasing competition between individuals and decreasing food supply (Jones and Kempton, 1978). However, the root dry weight is unaffected except at the highest nematode population densities, implying that a reduction in nematode numbers at high densities is associated with excess nematodes for the available food. Nicol (1991) noted similar trends, with a reduction in root dry weight and multiplication rate with 15000 P. thornei per plant, however this effect was only significant at25"C as opposed to 20'C. Nicol (1991) further suggested the damage at25"C on Warigal may be greater possibly ll5 Chapter 6 Population Dvnamics and Yield Relations in the Laboraton' because it is more favourable for nematode development than either 15 or 20oC. In addition the growth of the wheat host is less vigorous at 25oC compared with 20'C. The literature suggests that several foliar measurements are affected in the presence of P. thornei (Section 2.4). The evidence presented here and also by Nicol (1991) found neither the number of tillers per plant or the height of the plant were affected by P. thornei, although there was a suggestion for a reduction in height. Nicol (1991) found a stimulatory response to the number of nodals at low initial nematode densities, however the fact that nodal roots of wheat plants are greatly reduced in the presence of high P. thornei densities could be expected to have large effects on the final grain yield of wheat, particularly in drought conditions. However, although the number of seeds per wheat head was reduced with high populations of P. thornei, the number of heads per plant was found to increase, suggesting the developmental physiology of the wheat plant was acting in a compensatory manner, with the overall total foliar weight per plant unaffected by initial density. As with many of the other experiments conducted in this section, stimulation of growth for almost all growth variables occurs with the lower initial densities of the nematode (Chitwood and Buhrer, 1946; Chitwood and Feldmesser, 1948; Chitwood and Esser, 1957 and Peter, 196I; in Wallace, 1963). This is a com.mon occurrence with many plant parasitic nematodes. Although both the root and shoot dry weights were significantly affected the total dry weight was not. It is possible that the degree of variability, expressed as the SED in this instance, was too great between individual replicates to distinguish significant differences. Unfortunately, this variability is common with nematodes, particularly with the migratory forms, due to the differences in initial penetration between individual replicates which ultimately affect the subsequent multiplication and damage caused to the host. l16 Chapter 6 Population Dynamics and Yield Relations in the Laboratorv 6.5 General Discussion The four experimental sections covered in the Chapter have involved the use of different container size and, in most cases different soil types. The nematode inoculum was derived from the same method, however different cultures were used for each experiment and may have varied with respect to fitness thereby affecting pathogenicity. Because of the above variables extreme care should be taken when comparing results from these experiments. An example of different results is between Section 6.1, where a linear relationship was seen between multiplication rate and initial nematode density (Fig. 6.2), and the last experiment (Fig. 6.25). Possible explanations can be presumed to be associated with the different soil type, container size and possible intolerances of the two wheat cultivars, Machete and V/arigal. This infers the need for a greater understanding of the relation between the nematode, host and the surrounding environment. Nevertheless the results presented clearly show that P. thornei can affect many growth variables and also displays population dynamic models typical of those documented for other nematode species (Ch 2.9). ln most cases with roots and shoots, P. thornei was found to cause a stimulatory effect on growth at low initial nematode densities. This effect in general was found to decline with both increasing P. thornei density and time. The population dynamics of the nematode were found to vary between experiments, probably due to the different experimental technique used. rt7 Chapter 7 Field Populations Dynamics and Yield Relations Chapter 7 Field Population Dynamics and Yield Relations of P. thornei 7.1 Introduction The only Australian field population study of P. thornei was carried out by Pattison (1993) on wheat in New South Wales . P. thornei had little multiplication between June and September due to low temperatures. Nematode multiplication occurred toward maturity of the crop but was dependent on environmental conditions. As Van Gundy (1972) observed, predictions of disease potential are based upon knowledge of pathogen population dynamics and techniques for estimating economic thresholds (Section 2.8). Accuracy of predictions depends largely upon the reliability of the nematode assay data on which they are based. Wide variation in soil distribution of nematodes creates serious sampling problems (Barker and Nusbaum, 1971). Meaningful population studies are possible in pots and small field plots. However, large plots are generally inappropriate because populations are usually patchy and the multiplication rate decreases as the population density increases (Section 2.8). The average multiplication rate for a field as a whole may give little indication of what is happening in its parts (Jones and Kempton, 1978). Assay procedures are often too insensitive to measure populations prior to planting. Also the infectivity of the nematodes present is usually unknown. Thus, refinement of the sampling and assay techniques may well play a major role in determination of economic thresholds (Nusbaum and Ferris, 1973). The most convenient time scale to determine population dynamics in annual crops is one year, when Pi is the "initial" population density at planting and P¡ is the "final" population one year later (preferably at planting time in the following year to avoid I l8 Chapter 7 Field Populations Dynamics and Yield Relations problems of aggregation) (Jones and Kempton, 1978). Pratylenchus is capable of multiplying for several generations during a single season, therefore they spread only from plant to plant due to their small size and relative immobility. P. thornel is found randomly distributed at sowing time, but distributions of nematodes become aggregated following multiplication and development in the wheat crop (Pattison, 1993). Sampling plans should be custom made for particular situations, since the relationship between the number of sampling cores and the relative error changes in response to many factors, including nematode species, density, field size, crop and soil type (McSorley and Parrado, 1982). In addition, all techniques of extraction from soil yield only a fraction of the nematodes present. Soils differ and the techniques suitable for a light soil may be inadequate for a heavy soil. It is important, therefore, to estimate the population of nematodes by the consistent use of a particular technique. Determination of the population of P. thorner usually involves collection of soil cores from the upper 15-20 cm of the profile where roots are present. Eastwood et aL (1994) found more than 507o of the total P. thornei population below 30 cm in Victoria, while Peck er aL (1993) found that in order to accurately quantify P. thornei populations on the Darling Downs of Queensland, soils needed to be sampled to a depth of 120 cm. In the Barossa Valley of South Australia on clay based soils, S. Taylor (pers. comm.) found 78Vo of P. thornei in the top 20 cm of the profile. Similarly, SOVo of the closely related species, P. neglectus, were found in the top 15 cm of a sandy soil on the Eyre Peninsula (S. Taylor, pers. comm.). Understanding of population dynamics and crop yield reductions in South Australia is required in order to design possible management strategies to control P. thornei populations and keep them below the damaging threshold. P. thorn¿i is known to limit yield of wheat (Section 2.8). There has been no examination of the population dynamics of this nematode and the related yield loss in wheat under South Australian conditions. The work described in this chapter is an attempt to provide that information. 119 Chapter 7 Field Populations Dynamics and Yield Relations 7.2 Materials and Methods A study of the population dynamics of P. thorn¿i was conducted over a two year period on a field trial site at Tanunda, in the Barossa Valley (80 km north of Adelaide) in South Australia. Originally two field sites were chosen. However, while the Mallala site (also in the mid-north of South Australia) was sown for two years, it suffered a mouse plague in 1993 and a severe drought in 1994. The Tanunda field site was selected because the soil had high populations of P. thornei, there was low incidence of root diseases (Rhizoctonia spp., Gaeumannomyces graminis and Heterodera avenae, refet to Table 7.2) and the paddock was generally clean (weed free) with a known cropping history. 7.2.1 General Method The aim of the field trial was to determine the nematode density at which damage occurred. The first year of the trial included sowing 12 host species with differing degrees of susceptibility selected from work done in Chapter 5. These varieties were grown in replicated field plots in order to establish a range of initial densities for the second year of the trial. The cultivars were selected from the information presented in Section 5.1. The initial density (P¡ vear l) was determined in each plot at the start of the first season and subsequent grain yields in each plot were measured. The yield in each plot was then related to the initial density (P¡ year 1¡. Having established a range of initial P. thornei densities, in the second year one of three wheat cultivars was sown into randomly selected plots across the whole trial area. Once again P. thornei initial densities were determined in each plot, designated as (P¡ year 1) or as (P¡ vear 2) and the final grain yields in each plot were determined. In addition the following year the (P¡ ]ear 2) or (P¡ year 3) was also determined. The multiplication rate (Pi vear 2 lPiYear 1) could then be related to the range of host species sown in the first year, and the relationship between Pi year 2 and the three wheat t20 ChcLpter 7 Fiekl Ponu.l.ati.ons and YieLcL ReLation.s hosts considered fol the second year. Furthermo¡e, [he P1, ]ear 2 or Pi year 3 p¿5 determined at the beginning of the following season, theleby also enabling the relationships between multiplication rate (Pi Year 3 / p1 year' 2) and P, Yem 2 to be examined. 7.2.2 Sampling Device The rnethod of sampling is extremely important to ensure the accuracy of the populations extracted, The majolity of P. thontei at Tanunda wele located in the top 20 cln of the soil profile (S. Taylor', pers. comm.). Hence this layer of soil was sampled. A modified soil coring device was used based on the model descnbed by Thorrpson ¿/ a/. (1988), illustratedin Fig.7.1. Each core was taken by pushing the sampling device 20crninto the ground, rotating the handle 180', lifting the device out of the soil and t'etnoving the soil by hand into a collection bag. Fig.7 .1 The sampling device used to take soil cores fiom the two year trial conducted at Tanunda (1cm=6.5cm). 12l Chapter 7 Field Populations Dynamics and Yield Relations 7.2.3 Field Trial Layout The trial ¿uea was 80 m x 48 m, comprising 192 plots including 62 border plots. Each plot was 2 mx 10 m, with the sample area being 1.5 m x 8 m. The border plots in both years were sown with the wheat cultivar Janz. The trial was set up as a randomised complete block design (RCBD) in the first year. Ten replicates of each of the 12 varieties, plus a fallow treatment, were sown (Table 7.1, Plate 7 .1, Fig. 7 .2). Table 7.1 : The varieties, sowing rates and nematode susceptibilities used in the two year field trial at Tanunda. ¡o seeds/mZ sowing rate ncmatode kslha susceotibiliw lst Year l. Glenelg (Linseed) 67.5 45 low 2. Barossa ( Canola) 7.5 5 low 3. Grimrnett (barley) 115.8 200 77 low-mod. 4. Echidna(oat) 150 250 100 low-mod. 5. Yalloroi(durum) 130.5 200 87 low-mod. 6. Cunency (triticale) 138 200 92 mod. 7 . Tahara (triticale) t45.6 200 97 mod. 8. GS50A (wheat) t23.6 200 82 mod. 9. Molineux (wheat) 100.4 200 67 mod. 10. Machete (wheat) lll.3 200 74 high 11. Spear (wheat) rt7.6 200 78 high 12. Warigal (wheat) 118.7 200 79 high 13. Fallow B=border Janz wheat t20 200 79 unknown no. no seedslmz sowing rate nematode kglh susceptibilijr 2nd Year 1. Warigal (wheat) I 18.7 200 79 high 2. AUS4930 (wheat) 95 200 '19 low 3. GS50A (wheat) r23.6 200 82 low B=border Janz wheat r20 79 unknown Cløpter7 Field Ponulations Dvnamics and Yield Relations Plate 7.1: The field trial layout at Tanunda in the first year of the experiment, taken 5 months after sowing. Plots (2 x 10m) of the 13 different ffeatments were ¿uranged as a RCBD. Plate 7.2 : A representative picture of a lesioned wheat root system taken 5 months after growth. In this case the cultivar is Spear, showing dark brown lesioning on both the main seminal roots, extending to the finer lateral branches ( 6X magnification). 122 Chapter 7 Field Populations Dynamics and Yield Relations First Year (1993) 45678 I B B B B B B B B 2 B l0 l1 12 R lô I B 3 B ll R q 1 6 4 B ,) 4 B 1'.) l0 I c 5 B 5 B 3 4 '7 6 l0 7 B 6 B 6 5 5 l3 4 6 B 7 B 1 6 l3 ll 1 1 B I B 1? 6 1 l3 2 B 9 B q l? 2 t 1t B l0 B 2 l0 1) 5 J 12 B 11 B 9 l3 t0 1) ll B l2 B 4 7 5 , I 4 B ,7 13 B ll 4 4 ll 3 B t4 B 8 2 s 2 I 5 B 15 B 1? 4 11 6 B t6 B 7 I 1l 12 5 10 B t7 B 1) 12 2 5 6 l3 B l8 B 5 R 6 1 I s B t9 B 1 ll 10 I 1 1 B ,7 20 B 6 3 1 12 B 2t B lo 1 1? 13 ) B ,a B 1 4 R R l0 R B 23 B 9 8 t2 4 B B B 24 B B B B B B B B Second Year (1994) 123 45678 I B B B B B B B B a B ) I 2 ) B 3 B ) I 2 ) 1 B 4 B I I 2 1 ) B 5 B ) 3 I I ) 3 B 6 B 1 1 I I 2 I B 7 B ? I I 1 3 B 8 B 1 2 2 I 1 B 9 B 3 1 I I ) 1 B 10 B 1 I 1 I 3 B 11 B I 1 1 ) B t2 B 1 l I 1 l B 13 B ? 1 3 3 ) B l4 B ) 3 I I I B 15 B I 2 B .l t6 B 1 I 2 B t7 B 1 3 ) 2 2 B 18 B I l ? ) B t9 B I I 1 2 I B 20 B 1 3 2 ? ) 2 B 2l B I I I I I j B ,a B t I 3 ? 3 I B 23 B 3 2 2 2 B B B 24 B B B B B B B B rig7.2_: Layout of ranunda field trial for the 2 consecutive years. (refer to Table 7.I for numerical explanation of cultivars). 123 Chapter 7 Field Populations Dynamics and Yield Relations The following year, 3 wheat varieties were sown across the previous year's trial. The 3 varieties were allocated to individual plots on the basis of the previous year's rotation and initial nematode density, in order to give an even representation of both. Thirty plots each of AUS4930 (Section 5.2),the variety thought to be resistant to P. thornei, and the P. thornei resistant selection from Queensland, GS50A, plus 70 plots of a known intolerant, susceptible South Australian cultivar, Warigal, were sown (Table 7.1). The layout of the varieties over the two years is illustrated rnFig.7 .2. The various treatments applied to both trial sites over the two year duration of the trial are summarised in Table 7.2. The rainfall and temperature for Tanunda is presented in Fig. 7.4 and 7.5. Table 7.2:The various treatments to the Tanunda field trial site 1993 1994 ll3 varietal treetmentsì (3 v¡rietal treatments) Heterodera avenae (CCN) 0 U Rhizoctonia 0 0 sowrng date 2t6t9'3 r6t6t94 fêrtiliser application 130 kg/ha DAP at sowing 130kg/ha DAP at sowins spray reglme Grass (1218193) Grass (418193) Hoegrass@ (1.5llha); Puma S @ (2.0l/ha); all except oats. all plants. Glean@ ; Broadleaf (1418193) oats only Mecoban@ (2.8Llha); all plants. Broadleaf (2418193) Mecoban@ (2.8l/ha); Starane @ (500m1/ha); wheat, barley, oats. all plants. Bucrril MA @ (1.4 L/ha); Broadleaf + Grass(24111193) Linseed only Roundup@ (l5Oml/ha); Goal@ (l25ml/ha) Lontrel @ (300m1/ha); Pathways. Canola only Broadleaf + Grass (2418193) Roundup@ (150m1/ha); Pathways. Initial P. thornei density P¡ vear I taken 17l8l93 P¡ year 2 taken 2016/94 sampled Pi year 3 taken 6/5195 Root systems sampled 6nU93 9/tU94 pre-harvest I nal harvestecl 4tL2t93 r7/12/94 124 ChapterT Field Populations Dynamics and Yield Relations Fig.7.4t Monthly rainfall for Tanunda during 1993 and 1994, in comparison to the 25 year mean. (source: South Australian Bureau of Meteorology) too 90 80 70 RainfÀ.lt 1993 60 Rainfau 1994 Rainfau 19ó8-1993 Rainfall (mrn) so 40 30 20 lo o Jan Feb MúchAprtl Måy June July Aug. Sept. Oct. Nov. Dec. Month of t}le year Fig. 7.5: Monthly minimum and maximum temperatures at Tanunda for 1993 and 1994. (source: South Australian Bureau of Meteorology) 30 25 20 max temp 1994 min temp 1994 Temperature ( C¡ rs max temp 1993 min temp 1993 lo 5 o Jan Feb MarchApril May June July Aug, Sept. Oct Nov. Dec. Month of the Yea¡ 7.2.4 Sampling Methodology for Initial Nematode Density Determination of initial densities in each of the field plots was made at the beginning of each year (1993 P¡ Year I, 1994 Pi year 2 and 1995 P; year 3). Initial investigations to determine an appropriate sampling methodology were carried out. Before the first crop was sown in April 1993, eight individual soil cores approximately lm apart were taken to a depth of 20 cm using a soil corer (Fig. 7.1). Six plots across t25 Chapter 7 Field Populations Dynamics and Yield Relations the whole trial area were sampled. The total number of soil samples was 48. The number of P. thornei per 200 g of soil from each sample was determined using the Whitehead extraction technique (Section 3.2.I) over two days. The heterogeneity of the samples from the 48 cores was analysed using Hierarchical Analysis of Variance. The analysis gave total, within group (plot) and among group (plot and core) variation. The plot of residuals versus fitted values indicated that a logarithmic transformation was required. The analysis indicated that within plot variation exceeded the between plot variation. Approximately 4OVo of the variation could be accounted for between the 6 plots, while the remaining 60Vo was accounted for within individual plots. The inference from this was that sampling 8 cores within a plot (8 m x 1.5 m) was not sufficient to give a reasonable estimate of the number of P. thornei per plot. Hence it was concluded that more cores per plot were necessary. Due to limitations in time and man-power and the fact that 130 individual plots were required to be sampled, the number of cores were almost doubled from 8 to 14 per plot to improve the reliability of the count for each plot. This sampling method is illustrated in Figure 7.3. Cores were taken between the third and fourth, seventh and eighth seeding rows in each plot. The 14 cores sampled from each plot were then combined because the analysis of individual cores from a plot would yield little extra information, as the treatment effect was the major variable assessed. One 2009 representative sample was taken from the total pooled soil samples to extract nematodes, using the Whitehead tray method (Section 3.2.1) over two days. The same plots were sampled three years consecutively with sampling as close as possible to previous coring sites. The cores for estimation of initial densities were taken as early as possible, just after the break of the growing season (Table 7.2). Soil was collected when the soil was wer enough to allow sampling, as the heavy clay based soil texture prevented sampling when 126 Chapter 7 Field Pooulation.ç and Yield Relations dry. Before each coring, the corer was covered with talcum powder to enable easy removal of the sticþ soil. lOm length row 1.... row 2 row 3 * soil core rt€ :F :& {€ {< row 4 {< t€ l.5m width row 5. row 6.0- I .2m0.. ..L....2....3....4.... 5... .6....7 ..... 8. 8- I 0.0m. row 7 {€:F*:F:1.*{. row 8 row 9 row 10 Fig-..7.3 : Diagrammatic representation of the position of 14 individual sampling cores within a plot. The sampling method used in the first year was reassessed in the second year of sampling to confirm its reliability. From twenty-seven randomly selected plots, 14 paired cores from each plot were collected and combined to produce 2 composite samples for each plot. A 200 g representative sub-sample was taken from each sample and nematodes were extracted using the Whitehead tray extraction technique. Spearman's Ranking of Correlation Coefficient was calculated which indicated the pairs of samples were highly correlated with a t value of 5.0. This indicated sampling was consistent between each pair, and confirmed taking 14 cores from each plot was a reliable method of estimating density of nematodes. 7.2.5 Plant and Nematode Characters Measured 7.2.5.7 Plant Parameters Sampled Plant Samples Plants of different varieties were collected in both years of the trial, during the later pafts of the growing season (Table 7.2). The plots sampled were determined on the basis of the initial P. thornei densities calculated at the start of the season. In the first year, 3 plants were randomly sampled from both the highest and lowest initial density plots for all 12 varietal treatments tested (Table 7.1). During the t2'7 Chapter 7 Field Populations Dynamics and Yield Relations second year, 3 plants of the three different cereal varieties (4US4930, GS50A and V/arigal) were sampled from the 5 highest and lowest initial densities. Whole plants were dug out of the soil, the root systems were washed gently in water, and any symptoms of nematode damage were noted before the wet weight of the plants were recorded. The plant samples from each plot were combined and nematodes extracted using the Mister technique (Section 3.22). They were then counted and their numbers were recorded (Section 3.3). Ouantitative Growth Characters In the second year of the trial three plant growth characters were measured for each of the 5 highest and lowest initial density plots for the three different varieties. These characters were: maximum height of plants (cm), length of individual heads (cm) and number of tillers (including primary tillers). Ten individual plants per plot were selected visually to give a representative sample of each of the characters to be recorded. A plot was considered a single replicate. 7.2.5.2 Nematode Variables Sampled At the start of each season (1993, 1994 and 1995) the initial density of P. thorn¿i was determined using the sampling methodology described in Section7.2.4. 7.3 Results 7.3.1 Plant Parameters Sampled (Refer to Materials and Methods Section '7.2.5.1). Plant Samoles The data for samples of roots from both years were analysed separately using a RCBD. The two variables, P. thornei per plant and P. thornei per gram of fresh root, were investigated. The original analyses indicated heterogeneity of variance, so the data was log transformed, Logs (x+1), for both variables and years. t28 Chapter 7 Field and Yield Relations Table 7.3: ANOVA for the taken over the 2 of the field trial d.f.. ms vr Prob Year l: 1993 Variable : Log (P. thorneilplant+ \) Rep. I 4.459 Rep. Variety Stratum Variety 11 4.7431 2.39 0.082 Residual 1l t.982 Total 23 Va¡iable :Log. (P. thomeit g root + l) Reo. I 4.810 Rep. Variety Stratum Variety I I 4.194 2.t6 0.109 Residual 11 1.945 Total 23 Year 2: 1994 Va¡iable : Log (P. thorneilplant + l) Reo. 4 3.339 Reo. Varietv Stratum Variety 2 0.t20 0.05 0.949 Initial Density 1 22.288 9.7 t 0.006 Variety x Initial Density 2 8.202 3.57 0.048 Residual l9(1) 2,295 Total 28(1) Variable :Log (P. thorneil g root + l) Rep. 4 4.034 Rep. Variety Stratum Variety 2 2.303 0.92 o.417 Initial Density I 26.637 10.63 0.004 Variety x Initial Density 2 7.666 3.06 0.072 Residual 18(2) 2.505 Total 27(2) From the 1993 ANOVA (Table 7.3) there was no significant difference between varieties. Although non significant, the ranking of the 12 varieties from the first year is illustrated in Fig. 7.6. The ranking did not change irrespective of the units used (log P. thornei / plant + I or Log P. thornei / g root + 1). Visually, most of the wheat root systems showed lesioning and cortical degradation on nodal, lateral and seminal roots (Plates I .2,7 .3). The size of the wheat root systems at high initial densities was much reduced. This was particularly noticeable with Spear (Plate 7.3), Machete and'Warigal, three of the common South Australian wheat cultivars. r29 Chapter 7 Field Populations Dynamics and Yield Relations In contrast there was no visual difference in the size or degree of lesioning of the root systems of the two non-leguminous hosts, linseed and canola. The triticale, barley and oats showed some root lesioning, but little difference in the size of the root systems was seen at different initial densities (Plates 7.4,7.5). The second year of the trial revealed a significant effect of variety by initial P. thornei density. This is illustrated for log P. thornei per plant inFig.7.7. The numbers of nematodes between individual varieties were not statistically different. However, with V/arigal more P. thornei were extracted from the roots at high than at low initial densities. There is a similar trend for GS50A and AUS4930 although this is non-significant. Once again similar results were found for log P. thornei / g plant. 7.3.2 Ouantitative Growth Characters (Refer to Materials and Methods Section 7.2.5.1). Table 7.4 gives the ANOVA of plant growth characters measured for the three varieties (4US4930, GS50A and Warigal) during the second year of the trial. The data was analysed as a CRD. None of the variables showed significant interaction between variety and nematode density. However, in all cases a varietal effect was evident for height of plant (Fig. 7.8), head length per plant and the number tillers per plant (Fig. 7.9). In all cases the unadapted landrace AUS4930 had significantly greater growth than either GS50A or Warigal. 130 Chapter 7 Field Populations Dynamics and Yield Relations Fig. 7.6 : The number of P. thornei extracted pre harvest from the cultivars/varieties used in the first year of the Tanunda Field Trial (note: non significant). 8 7 SED = 1.4 6 c€ E- '0t 5 oltst Àl:l 4 oÞo -l 3 2 0 ËËËËÊEåEËÊ'ËË Fig. 7.7 : The interaction between variety and initial density on the numbers of P. thornei extracted from the 3 wheat cultivars pre harvest during the second year of the Tanunda Field Trial. 8 7 I highinitial densiry S ìow initial density 6 sED = 0.96 ñ Ë 5 I 'Er 4 tst rlol .t Àl 3 bo I 2 0 AU54930 GS5OA Warigal 131 Chapter 7 Field Populations Dynamics qnd Yield Relations Fig. 7.8 : The significant varietal differences in wheat cultivar height measured pre harvest during the second year of the Tanunda Field Trial. 80 nus¿s¡o 70 I E cssoA I Warigat 60 SED = 1.3 o ?50 ã(Ë o40 Þo -g 30 20 l0 0 vanety Fig. 7.9 : The significant varietal effect on the number of heads and number of tillers per plant fol lhe 3 wheat cultivars measured pre harvest during the second year of the Tanunda Field Trial. l0 9 I AUS4930 E cssoA 8 I Warigal 7 SED (no. heads per plant) = 0.22 0) SED (no. tillers per plant) = 0.36 C) E 6 (Ë (É Þ- 5 3 o 4 3 2 1 0 no heads perplant no. tillers per plant Chapter 7 Field Populations Dynamics and Yield Relations Plate 7.3 : Three representative Spear wheat roots from both low initial density plot (30 P. thornei per 2009 OD soil) and the high initial density plot (1506 P. thornei per 2009 OD soil), taken from the first year of the field trial after 5 months of growth. Evidence of more robust tillering capacity and root density is evident with the lower initial nematode density. However, severe restriction of both seminal and lateral root systems is seen with high P. thornei densities. Plate 7.4 : Thee representative Currency triticale roots from both low initial density plot (12 P. thornei per 2009 OD soil) and the high initial density plot (535 P. thornei per 2009 OD soil), taken from the first year of the field trial after 5 months of growth. The growth of the root system shows no apparent difference with low or high initial nematode populations. Plate 7.5 : Three representative Yallaroi durum wheat roots from both low initial density plot (27 P. thornei per 2009 OD soil) and the high initial density plot (2938 P. thornei per 2009 OD soil), taken from the first year of the field trial after 5 months of growth. The growth of the root system shows no apparent difference with low or high initial nematode populations. /.- Low HIGH INITIAL DENSITY INITIAL DENSITY SPEAR WHEAT li LOW HIGH INITIAL DENSITY INITIAL DENSITY CURRE,NCY TRITICALE \ HIGH LOw DENSI'TY INI'IIAI, DBNSI'TY INITIAI, YALI,AROI DURUM t32 Chapter 7 Field Populations Dynamics and Yield Relations Large differences were observed (visual assessment) between the varieties in the second year of the field trial. Warigal suffered extreme cortical degradation and lesioning at high initial densities of P. thornei (Plate 7.6). Plate 7.9 shows the extensive cortical lesioning associated with reduction of the main root system (seminal and nodal roots) and also the lack of the finer lateral branching on the seminal root system. The P. thornei resistant wheat cultivar GS50A showed some lesioning and reduced root volume (Plate 7.7),but not to the extent of Warigal. In contrast, AUS4930 (Plate 7.8), also thought to be resistant to P. thorn¿¿, showed little evidence of nematode attack or reduced size at either low or high densities. Table 7.4: ANOVA of three plant growth characters measured on AUS4930, GS50A and V/arigal in the second year of the field trial at Tanunda. m,s v.r Prob Yeat2:1994 Variable : Plant Height (cm) Plot Variety 2 7683.08 88.64 <0.001 Density 1 66.74 0.77 0,389 Variety.Density 2 29.28 0.34 o.7t'7 Residual 24 86.68 Plot.Plant 270 35,45 Total 299 Va¡iable : Head Length per plant (cm) Plot Variety 2 132.2036 55.27 <0.001 Density I 1.3736 0.57 0.456 Variety.Density 2 0.7906 0.33 o.722 Residual 24 2.3921 Plot.Plant 270 0.8059 Total 299 Variable : Number tillers per plant Plot Variety 2 554.54 85.01 <0.001 Density 1 L47 o.23 0.639 Variety.Density 2 8.49 1.30 0.29t Residual 24 6.52 Plot.Plant 27O 3.38 Total 299 Chapter 7 Field Ponulations Dynamics and Yield Relations Plate 7.6 : A representative Warigal wheat plant from both a low initial density plot (39 P. thornei per 2009 OD soil) and a high initial density plot (9044 P. thornei per 2009 OD soil), taken from the second year of the field trial after 5 months of growth. With high nematode populations there is evidence of extensive dark brown cortical lesioning accompanied by cortical degradation on the seminal root system. Reduction of the finer lateral branches at high nematode densities is also evident. Plate 7.7 : Arepresentative GS50A wheat plant from both a low initial density plot (61 P. thonnei per 2009 OD soil) and a high initial density plot (9416 P. thornei per 2009 OD soil), taken from the second year of the field trial after 5 months of growth. With high nematode populations there is evidence of extensive dark brown cortical lesioning accompanied by cortical degradation on the seminal root system. Reduction of the finer lateral branches at high nematode densities is also evident. Plate 7.8 : A representative AUS4930 wheat plant from both a low initial density plot (11 P. thornei per 2009 OD soil) and a high initial density plot (2450 P. thornei per 2009 OD soil), taken from the second year of the field trial after 5 months of growth. The plants show no visual evidence of lesioning or apparent differences with root growth at different initial nematode densities. LOW HIGH WARIGAL LOW HIGH GS5OA LOW HIGH AUS 4930 Chapter 7 Field Populations s and Yield Relations Plates 7.9 : Three representative'Warigal wheat roots from low (39 P. thornei per 2009 OD soil, TOP), moderate (4752 P. thornei per 2009 OD soil. MIDDLE), and high (9044 P. thornei per 2009 OD soil, BOTTOM) initial density plots taken from the second year of the field trial after 5 months of growth. The degree of root lesioning on both seminal and lateral root as well as the reduction in both the seminal and finer lateral root system was accentuated by increasing nematode density. The loss of grain yield between low and high plots was 27Vo. t I .* í ,l ( ,l I /t, I I I ( 133 Chapter 7 Field Populations Dvnamics and Yield Relations 7.3.2 Nematode Variables Sampled (Refer to Materials and Methods Section 7.2.5.2). r993 The initial density from the first year of the field trial (Pi year I ) for each individual plot was regressed against the yields of the respective cultivars (Table 7.5). Machete and Spear were the only two varieties which indicated a significant linear relationship between initial density and yield (Fig 7.10), with increasing nematode density correlated with a decrease in the yield of wheat. Table 7.5: Regression analyses between initial P. thornei density and yield of various wheat cultivars used in the 2 field trial at Tanunda. m.s v.r Year 1: 1993 Variety: Spear Response variable : Yield Fitted terms : constant, Pi Year I Regression I r.2714 5.4 Residual 8 0.2356 Total 9 0.3507 F 1,9 tables 5.32 < v.r. = implies linear relationship is just significant Variety :Machete Response variable : Yield Fitted terms : constant, Pi Year I Regression I 0.82470 36.16 Residual 7 0.0228t Total 9 0.t2304 F 1,7 tables 5.59 < v.r. = implies linear relationship is strongly significant Year 2: 1994 Variety: Warigal Response variable : Yield Fitted terms : constant, Piyear 2 Regression I 0.44833 5.49 Residual 65 0.08164 Total 66 0.08719 F 1,65 tables 4.00 < v.r. = implies linear relationship is just significant 134 Chapter 7 ield Populations Dynamics and Yield Relations The visual differences between plots of Machete are illustrated in Plates 7.10, with denser growth and heading at low initial P. thornei density relative to the high nematode density plot. 1994 An ANOVA was conducted comparing the yields of the 3 wheat varieties used in 1994 trials in order to determine whether the previous rotational crops used in 1993 had any influence on the results for the second year. For each of the three 1994 wheat varieties there was no indication that the 1993 rotation treatments had an effect on the observed yield. Given this result, a regression analysis was conducted between initial P. thornei density for the second year of the trial (Pi year 2) against the individual plot yields for 4US4930, GS50A and V/arigal (Table 7.5). Neither of the resistant wheat yields had a statistically significant relation with the initial P. thornei density. Warigal, like Spear and Machete in 1993, had a significant linear relationship with yield decreasing as initial nematode density increased (Fig. 7.11). A further regression analysis of initial P. thornei density for 1993 (Pi year 1) against the initial P. thornei density for 1994 (Pi year 2 s¡ p¡year l) was carried out for all 3 varieties (4US4930, GS50A and Warigal) used in the second year of the trial (Table 7.6). Both the resistant varieties AUS4930 and GS50A were found to have a significant linear relationship between initial and final P.thornei density (Fig. 7.12). However, a quadratic relationship was found to be more appropriate for'Warigal than a linear (Fig.7.I2). It could be that the larger number of observations for Warigal results in increased power when testing the quadratic model versus the linear. Thus the relationship may be quadratic but for AUS4930 and GS50A where there is a much smaller number of observations the linear relationship is not demonstratably inferior to the quadratic. The relationship between the initial P. thornei density in the 1994 (P¡ year2) and the multiplication rate ( Pi vear 2 / Piyear l) was examined (Fig. 7.13). At low initial 135 Chapter 7 Field Populations Dynamics and Yield Relations nematode densities, the multiplication rate is high, but higher initial P. thornei densities are associated with reduced multiplication. No statistical analysis was conducted to test association as the Y axis (multiplication rate) is highly dependent on the X axis (Pi lear 2). Fig. 7.10 : The relationship between the initial density of P. thornei andthe grain yield of the two wheat cultivars, Machete and Spear, grown in the first year of the fiõt¿ triát. 6.O 5.5 t 5.0 4.5 t t ¡ . Machele Wheat Yield 4.O I Spear (t/ha) a 3.5 a I 30 2.5 2.O 0 2000 3000 4000 Initial P. thomei Density Fig. 7.11 : The relatiglslip between the initial density of P. thornei and,the grain yield of the wheat cultivar warigal grown in the second year of the f,reld trial. 2.50 2.25 f 2.OO T Yield Warigal (/ha) r.75 1.50 t.25 1.00 0 2000 4000 6000 8000 10000 Initial P. thornei density (2009 OD soil) 136 Chapter 7 Field Populations s and Yield Relations Fig.7.1?: The relationship between the initial and final density of P. thorn¿l under three different wheat hosts grown in the second year of the field trial. o 25000 Ø Y(4US4930) = 1326 + O.27 5xi + 22500 Y (GS50A) = 1l l0 + 0.375xi o Y(Warigal) =912 + 3.882xi -O.NO2523xi2 bo 20000 . AUS4930 cì 17500 *** o GS50A Ø 15000 + Warigal (l) t2500 c¡I + 10000 + olEI ;t 7500 ++ cÉ 5000 f¡. 2500 a 0 0 2500 5000 7500 10000125001s0001750020000 Initial P. thornei density (2OOg OD soil) Fig. 7,1_3 :_The relationship between the initial nematode density and the multiplication rate of P. thornei under the wheat cultivar Warigal, grown in the second year ofihe field trial. 60 55 50 45 40 35 P. thornei Mul--nptication rate 30 t 25 20 l5 a l0 a a 5 f a a. 0 0 5000 10000 1s000 20000 25000 30000 Initial density!¡@!(200 g OD soil) Chapter 7 Field Pooulations Dvnamics and Yield Relations Plates 7.10 : A low initial density plot (42 P. thornei per 2009 OD soil, BOTTOM), and a high initial density plot (3127 P. thornei per 2009 OD soil, TOP) of Machete taken from the first year of the field trial after 5 months of growth. The plot with few nematodes (BOTTOM) shows evidence of denser tillering and head production in comparison to the plot with many nematodes (TOP), which in comparison is less dense and relatively unthrifty. 137 Chapter 7 Field Populations Dynamics and Yield Relations Table 7.6: Regression analyses between 1993 initial P. thornei density (Pi year I ) and 1994 year initial P. thornei density (Pi vear 2 ) from the second year of the Tanunda field trial. d.f. m.s. v.r Year 2: 1994 Variety: AUS4930 Response variable 2 Pi Year 2 Fitted terms : constant, Pi Year I Regression I 12278014 14.66 Residual 27 836982 Total 28 1245591 F 1,27 tables 4.21 < v.r. implies significant linear relationship Variety: GS50A Response variable .PiYear 2 Fitted terms : constant, P¡ Year I Regression 1 5991r32 6.78 Residual 26 883906 Total 27 1073062 F 1,26 tables 4.23 < v.r. implies signifrcant linear relationship Variery : Warigal Response variable . PiYear 2 Fitted terms : constant, Pi Year I Regression I 6.7478+08 42.30 Residual 68 1.595E+07 Total 69 2.5508+07 F 1,6g tables 4.00 < v.r. implies significant linear relationship Response variable z Pi Year 2 Fitted terms : constant, P¡ Year l, 1t; vear l;2 Regression 2 4.817E+08 40.50 Residual 67 1. I 88E+07 Total 69 2.5508+07 F 1,67 tables 4.00 < v.r. = implies significant quadratic relationship Variance Ratio was calculated to determine which model is more appropriate (linear or quadratic), i.e. to test the null hypothesis Br=Q. F 1,67 tables 4.00 < v.r. calculate d (24.04), therefore implying the quadratic model more appropriate. 7.4 Discussion The results presented here confirm that P. thornei is not just damaging to wheat in aseptic laboratory tests (Nicol, I99I, Ch 6), but also in the cereal growing regions in South Australia. The manipulation of P. thornei numbers in the field using a range of cereals and non-leguminous hosts was successful in producing a range of initial P. thornei 138 Chapter 7 Field Populations Dynamics and Yield Relations densities. These were used to examine the damage relations on wheat as well as the population dynamics of the nematode. Elston et aI. (1991) used a range of resistant and susceptible potato genotypes to manipulate population densities of Globodera pallida in a field in Scotland. Use of biological means to create a range of initial densities was found to provide a useful alternative to the application of chemicals (nematicides) or addition of a range of densities prepared from cultured inoculum. There was no indication of separate effects of the 13 rotations on yield performance in 1994. Although the primary aim of the first year (1993) of the field trial was to manipulate nematode numbers, P. thornei was found to reduce yields significantly for two commonly cultivated susceptible wheats, Spear (38Vo reduction with 1506 P. thornei 12009 OD soil) and Machete (27Vo reduction wtth3127 P. thornei l2OOg OD soil). It is possible that similar trends may have been expressed for some of the other cereals investigated, however the naturally occurring range of initial densities may not have been high enough to make this apparent and as there were only 10 pairs of values for each regression. That is, application of the regression where there was only 10 plots for each variety plus relying on the natural range of initial densities present was conducive to providing a powerful statistical test of the 12 varieties used(Appendix B). The second year (1994) of the trial, showed that the South Australian wheat \Warigal is highly susceptible to and intolerant of P. thornei with yield losses of 3Vo at low initial P. thornei densities (1000 I 2O0g OD soil) and 277o at higher initial nematode densities (9000 P. thornei / 2OO OD soil). This regression was more powerful than that for Machete and Spear in 1993, as it was based on 70 individual regression points. Thus while Machete and Spear suggested a linear relation it would appear that the true relationship may be quadratic but to establish this we need considerable observations given the large experimental error associated with field data. t39 Chapter 7 Field Populations Dynamics and Yield Relations Thompson (1993) suggested that P. thomei populations exceeding 500 nematodes / 2009 OD soil in the upper layer of the soil in Queensland was the economic threshold for intolerant wheats. Work by Taylor and McKay (1993) in South Australia with the use of the nematicide Temik@ showed that populations of 750 P. thornei / 2OOg OD soil resulted in yield losses of up to70Vo. Initial P. thornei populations of 500-1000 P. thornei / 2O0g OD soil in New South Wales indicated potential nematode problems could occur, while populations greater than 1000 / 20Og OD soil posed a serious economic threat (Pattison, 1993). The initial P. thornei densities and the associated significant yield loss of the wheat cultivars Warigal, Spear and Machete from the two year Tanunda field trial indicate economic thresholds which differ from those previously forecasted. V/ith respect to Tanunda trial area over the two year period, the density threshold for economic damage was much greater with V/arigal in the second year of the trial than either Machete or Spear in the first year. This may reflect differences in the tolerances of the varieties or may be associated with the seasonal differences (possibly rainfall) at the trial site between the two years investigated. However, work by Pattison (1993) suggests the economic threshold for damage in dry seasons will be lower, given that nematode damage to the roots may interfere with water uptake. It is important to note that sampling nematode populations in the soil is associated with an array of potential sources of variation arising from soil collection, transport and storage, extraction and counting (Dropkin,.1989). With the technique used in this field experiment, most soil cores are obtained from the upper regions of the soil surface (0- 20cm) where both soil and roots are present. Cores are usually combined, mixed and subsamples taken for extraction and counting. Dropkin (1989) notes that the actual count determined is only 2O-25Vo of the "true mean" of the population. In combination with the above factors involved with the sampling, the numbers of plant parasitic nematodes in the f,reld situation are determined by the carrying capacity of the environment or the available 140 Chapter 7 Field Populations Dynamics and Yield Relations resources (Ferris and Wilson, 1987). Resource availability refers to the abundance and state of the current or previous food resource (determined primarily by its physiology, activity and health), its distribution within the soil matrix (both horizontal and vertical) and its host status to the nematode. The experimental situation, size of the wheat field, variability in soil type, topography, cropping history and the man power available will determine what sampling technique is utilised. As previously discussed in northern NSV/ the distribution of P. thornei is more evenly dispersed than for other nematode species in other crops (Pattison, 1993), which allows sampling procedures to be less intensive than those recommended for aggregated (or patchy) nematode species in intense agricultural or horticultural crops. Pattison (1993) also suggested aggregation of P. thorn¿i was close to uniform at the time of sowing, but became aggregated later in the season. This supports the method of sampling used here. Although samples were taken as close to the start of the cropping season as possible when P. thornei populations should be uniformly distributed, particular parts of the trial paddock at Tanunda showed aggregations of nematodes. These were not associated with different rotational combinations as the cropping history of the paddock was known. They must be attributable to some form of environmental and/or edaphic influence, although Pattison (1993) could not correlate such aggregations to a plant or edaphic factor. This phenomenon is not uncommon (Jones and Kempton, 1978), and many species of plant parasitic nematodes may have an overall light infestation interspersed with a few patches of high density. Use of average population densities may suggest that the multiplication is small, but this may be misleading. Estimates of densities from such field trials as Tanunda generally have proportionally high variance compared to their means, confirming the patchy distribution (Wallace, 1963). Here, the frequency distribution of P. thornei initial densities is always skewed to the left, with the lower counts more numerous than high, and the bell-shaped distribution consistent with normal t4l Chapter 7 Field Populations Dynamics and Yield Relations distributions seldom if ever seen. Given that field populations of nematodes are not universally uniform, the method of sampling and interpretation of data is of paramount importance. As a result of the skewed initial P. thornei densities found in both years of the Tanunda trial, the subsequent regressions of density and yield for both years are not strong. This is so particularly for the first year, where the regression was only based on 10 data values. In the second year, where there were over 60 data points for Warigal and 30 each for AUS4930 and GS50A, the statistics are more reliable, however it could be that the statistical outcome has more to do with the number of data pairs than a fundamental difference between V/arigal and the other two varieties. Crop rotation is known to be the single most useful practice of nematode control and is effective if few hosts are grorwn often. That is, the ability of specific susceptible crops to multiply P. thornei will determine the number of resistant varieties which need to be grown in succession in order to reduce nematode populations to below the economic threshold for damage. More care should be taken to quantify differences in P. thornei populations between alternative crop species under field conditions (Thompson, private conìm. 1993). Although the primary aim of the first year of the trial at Tanunda was to manipulate a range of initial P. thornei densities, the use of 12 different rotational crops offered some information about the relative susceptibilities of these crops. Even though non-significant, there was a trend for the wheats to multiply greater numbers of P. thornei than the triticale, durum and non-leguminous hosts. Work by Lawn and Sayre (1992) in Mexico similarly found greater P. thornei multiplication on bread wheat than either durum or triticale varieties tested. The closely related species, P. pratensis was increased more by wheat and less by oats and barley, and least by rye (Oostenbrink er al., 1956). Thompson (private comm. 1993) found in Queensland field populations of P. thornei on linseed were much smaller than on wheat. In NSW, Pattison (1993) tested a selection of cereals and found a range of resistance to P. thornei. The barleys Skiff and Grimmett did not increase nematode populations, and the durum Kamilaroi had excellent resistance. 142 Chapter 7 Field Populations Dvnamics Yield Relations The ranking of varieties from the first year of the fielcl trial (Fig. 7.6) showed a ranking of susceptibility to P. thornei consistent with that observed in screening techniques used in the laboratory (Ch. 5). Similar studies with the potato cyst nematode G. pallida on potatoes showed that pot tests could be directly correlated to field performance (phillips, 1984). Once the relationship between performance of the standards in the pots and the field is known, it should be possible to predict the effectiveness of a variety in the field from pot tests (Phillips, 1985). Phillips (1984) also found that when estimaring rhe resistance of a partially resistant potato clone, it was preferable to rank clones consistently in order of resistance rather than use absolute values on nematode multiplication rates. Resistant standa¡ds should be included because without reference to these the results of pot tests may be misleading (Phillips and Trudgill, 1985). por tests have advantages over field trials as they are more convenient, less effort, the growth conditions can be standardised, the inoculum and other environmental factors are easily manipulated for proper replication, and initial and final numbers can be more accurately measured than in the field (Phillips and Trudgill, 1985). It is thus more likely that statistically significant results can be obtained from laboratory studies. However, laboratory results should always be verified by field evaluation. There is little information on the relation between population density and the increase in numbers of Pratylenchus spp. (Wallace, 1963). The opportunity to measure the initial p. thornei density over a two year time frame in the Tanunda field trial allowed investigation of the field population dynamics of the nematode. The relationships between initial and final density and the initial density and subsequent multiplication rate of P. thornei were demonstrated for the three wheat cultivars assessed in the second year of the trial, and conform to the models previously described for other plant parasitic nematodes (Section 2.e). r43 Chapter 7 Field Populations Dynamics and Yield Relations Both tolerance and resistance are linked to the population curve relating initial and final densities of nematodes (Fisher, 1993). The results of the field trial showed that in a susceptible cultivar such as'Warigal, the multiplication rate is high (up to 20-30 times) at low initial densities, but dropped rapidly reaching unity, at which time the population merely replaced itself, as initial density increased. This level is commonly referred to as "equilibrium density". With P. thornei, the equilibrium density and associated multiplication rate depend on external conditions as well as the inherent characters of the nematodes and host plant (Seinhorst, 1967). Unfortunately, the influence of these may not necessarily be related and a change in conditions may result in an increase of one and a decrease ofthe other or in increase or decrease ofboth (Seinhorst, 1967). In this trial the equilibrium density of P. thorn¿i on the susceptible host Warigal occured at an initial nematode density of about 10,000 / 2O0g OD soil. Pauison (1993), found the equilibrium density for initial nematode density was approximately 2000 /2009 OD soil, with populations above this leading to a decline in nematodes present in the soil at harvest. Seinhorst's (1967) work in the Netherlands found P. crenatus on cereals reached a maximum equilibrium density of 2400 nematodes /2}Ogsoil. At Tanunda, the equilibrium density for P. thornei with Warigal was well above examples previously documented. However, Seinhorst (1967) showed that the equilibrium density of p. crenatus was consistently different in different fields with the same cropping sequences during seven sequential years of observation. This suggests that in order to gain an understanding of population dynamics of a nematode several seasons of data are required so that future predictions are based on averages. As previously discussed (Section 2.8), control of nematodes is primarily about control of crop damage. The damage caused by root feeding nematodes is proportional to their population density (Seinhorst, 1965). In order to control nematodes, information about the damage threshold and the associated strategies to decrease populations to below densities threatening the next susceptible crop are necessary. In the trial described here, t44 Chapter 7 Field Populations Dynamics and Yield Relations the distribution of the data points was concentrated at the lower region of initial p. thornei densities and there was limited data to fit the models described in Section 2.8 or by Elston et al. (199I). Instead of log linear, exponential and inverse linear models being applied only linear and lower order polynomials were considered. In the Tanunda trial, the Warigal yield loss in relation to the initial P. thornei density was defined by the simple linear regression equation; Yield Reduction = 1.8617-0.0000556 x (initial p. thornei density). For example, if I\Vo yield loss was considered economically unacceptable (2.42tlhato 2.I9t/ha), then from the regression P. thornei populations need to be less than 5,905 l20Og OD soil ro avoid such a yield penalty. Most nematologists use resistant plants in order to maintain nematode populations below the economic threshold for damage. As discussed (Secti on 2.9), at present there are no varieties known to be resistant to P. thornei within the Gramineae oÍ the Leguminosae, the two most common plant families associated with rotational wheat growing in South Australia. However, the response of the two putative resistant wheats tested at Tanunda suggests they have potential. Unlike Warigal, which was found to have a quadratic relationship between initial and final P. thornei densities, both AUS4930 and GS50A had simple linear regressions. From the regression equations, it can be seen the rate of population increase over the range of initial densities between 0 - 15,000 p. thorneil211g OD soil is 0.375 for GS50A and 0.2755 for AUS4930 compared with 3.882 for Warigal. Considering the hypothetical example above, populations at the damaging density would be effectively reduced with both GS50A and AUS4930 from 5905 to 3324 for GS50A and2953 for 4US4930. This further suggests thar both AUS4930 and GS50A have field resistance to P. thornei . As with Heterodera avenae in South Australia (Fisher and Hancock, 1991), the resistance for P. thornei will not avoid damage entirely. Use of resistance will not eradicate the nematode, which will remain a potential th¡eat should susceptible cultivars be grown too often. 145 Chapter 7 Field Populations Dynamics and Yield Relations The trends observed in the field population dynamics are similar to those described by many nematologists (Section 2.8). At low initial densities of the nematode, multiplication can be up to 30 times. As the initial density increases, a corresponding decrease in multiplication rate is observed (Fig. 7.13). Multiplication rate is affected by factors such as soil temperature, soil type, soil moisture as well as initial density of the nematode (Phillips, 1985). Thus, although a resistant variety may lower nematode populations, due to the polycyclic nature of P. thornei, associated with very high multiplication at low initial densities, the effectiveness of this resistance is less where it is used to control monocylic nematodes. As a consequence studies such as these are imperative in order to forecast the effectiveness of the resistance. Even though the data presented is not statistically strong, it illustrates how mathematical relationships can be used to predict the effect of P. thorn¿i on wheat yield. As with the extensive work by Elston et al. (1991) with potato cyst nematode, combining such models with one for estimating the population dynamics of nematodes would provide a powerful means of developing integrated control strategies utilising resistant cultivars for the manage ment of Pratylenchus. AUS4930 and GS50A gave evidence of field resistance from the initial and final densities measured over the second year of the field trial. However, plants sampled relatively late (at grain filling in November) in the growing season (with numbers of p. thornei in the roots counted) produced atypical results (Fig.7 .7). At that time, there was no significant difference in nematode numbers between varieties. The highly susceptible wheat Warigal had similar numbers of nematodes to the resistant wheats. It is possible that the resistance mechanism in AUS4930 and GS50A may act very late in the growth of the wheat plant. Again, the seed populations may not have been completely homozygous for P. thornei resistance, particularly with the landrace AUS4930. Further, the initial densities between the different plots sampled for the three varieties may not have been truly comparable, hence confounding the corresponding multiplication rate measured. In t46 Chapter 7 F ie ld Populations Dvnamics and Yield Relations addition, although most roots were collected, the heavy soil type precluded effective sampling of the whole root system, particularly by this late (dry) stage in the season . The data also suggested that Warigal had significantly more nematodes in the roots in the high initial density plots relative to the low initial density plots sampled which would question the theory of reducing multiplication rates over a range of initial nematode densities. The confusion caused by such data further supports the findings of Jones and Kempton (1978), who suggest assessing population changes at the start of the season and again one year later. Most of the symptoms produced by P. thornei were comparable with those described by other workers (Section 2.4), although like most field work they were not found to be a statistically significant difference between few and many nematodes. Van Gundy et al. (1974) also found suggestions of stunting, reduced tillering and occasionally reduced head length. It was of interest that the resistant wheat, AUS4930 had much greater tillering capacity and head length. This may be of agronomic advantage for breeding than the current commercial varieties, although the excessive height may make it more prone to lodging. The histopathological symptoms produced by P. thornei arc similar to those previously reported (Section 2.3.2). Cortical degradation was indicated by the visual appearance of lesions on the root system, and was more extensive on susceptible wheats such as Warigal, Spear and Machete than AUS4930 and GS50A. Barley, triticale and non leguminous hosts showed less cortical degradation. This was similarly found in laboratory screening work (Chapter 5). As reported by Baxter and Blake (1968), segments of the cortex were found to slough off and expose the stele. Microscopic investigation similarly showed, P. thornei distributed in the cortical cells of the root system, with adults and eggs usually situated in the cortex parallel to the long axis of the root. Plants gro\¡/n in the high initial density plots, particularly the wheats highly susceptible to P. thornei,had a severe degree of cortical degradation. Work by Simmond 147 Chnpter 7 Field Populations Dynamics and Yield Relations and Sallans (1933) and Sallans (1942) showed loss of seminal roots significantly reduced the grain yield with the loss of the nodal roots eccentuating this damage. As a consequence, the damage to wheat caused by P. thornei during relatively dry season would be expected to be greater than in wet seasons. In conclusion, the evidence indicates that P. thornei reduces yield on wheat in the field. Most of the commonly cultivated South Australian wheats appear highly susceptible while AUS4930 and GS50A suggested resistant wheats in the field did show resistance. Studies on the population dynamics of P. thornei associated yield with nematode multiplication relationships, give the f,rrst opportunity to understand the field behaviour of P. thornei. These results provide some preliminary field evidence of the control management opportunities which may exist using rotational combinations. 148 Chapter I Resistance Response in Cereals Chapter I Plant Genetic Control and Possible Mechanisms of P. thornei Resistance in Cereals 8.0 General Introduction Plant resistance has been identified as the major priority in attempts to control nematodes, over chemical, biological, cultural and regulatory control (Roberts, 1990). Resistant varieties maximise and stabilise yields through their effects on nematode population dynamics (Cook, 1974). A plant is resistant when the ability of the nematode to feed, develop and reproduce is inhibited ('Wallace, 1963). Breeding plants with disease resistance relies upon genetic variation in the host plant population exposed to the parasite (Tinline et a1.,1989). These interactions are unique and the inheritance of the resistance relates to a specific pathosystem. Plant resistance has been found and developed for highly specialised plant parasitic nematodes such as Globodera, H etero de ra, M eloido gyne, Rotylenchus, Tylenchus and Ditylenchu.r which (with the exception of Ditylenchus) have a sedentary endoparasitic relationship with their host (Roberts, 1990). Nematodes which feed ectoparasitically or are migratory endoparasites are less likely to induce resistance due to the transient nature of their feeding. However, several plant species includingtea (Camellia sinerisis l. ), coffee (Coffea liberica I.) and tobacco (Nicotiana tabacum L.) have resistance to Pratylenchøs (Dropkin, 1989). In commercial agriculture, single genes for resistance are a priority, as most plant breeders have problems breeding successfully with multiple gene controlled resistance. In addition to the mode of inheritance of the resistance, the mechanism of the resistance process also determines the field applicability of this source of resistance (Cook, I974). t49 Chapter I Resoonse in Cereals With sedentary nematodes in particular there are numerous reported cases (Howard and Cotten, 1978 and Cook and Evans, 1987) which support Flor's gene for gene hypothesis (Flor, 1956), i.e., for each gene controlling the response in the host plant there is a specific and complementary gene controlling pathogenicity in the pathogen. This chapter is divided into two sections. The first describes a preliminary investigation of the mechanism of the resistance and the second an attempt to determine the genetics of P. thornei resistance in the wheat variety,4US4930. 8.1 Initial Penetration of Resistant and Susceptible Hosts 8.1.1 Introduction The ways that plants defend themselves against attack by nematodes are varied. Each way is usually specific for a given pathogen-host relationship and depends upon the activation of a coordinated multicomponent defence mechanism (Dropkin, 1989). Dropkin (1989) refers to four possible modes of plant resistance: barriers to attraction, reduced rate of nematode growth in an incompatible host, hypersensitivity response of plant cells penetrated by nematodes, and/or inhibition of growth of sedentary females. As previously described from the laboratory screening assay (Section 5.2,5.3) and also from the field data (Ch. 7), the wheat genome (Triticum aestivum) offers a range of susceptibilities to P. thornei . The mechanisms for resistance to P. thornei are unknown. This experiment was an initial attempt to determine whether reduced initial penetration with identified resistance lines was a major factor contributing to host resistance. The highly susceptible wheat cultivar Machete and two resistant wheat lines GS50A (selection from Queensland) and AUS4930 (from the Australian V/inter Cereals Collection, originally from lraq) were used. Information on the mechanism of resistance is of paramount importance, particularly in South Australia, because if the 150 Chapter 8 Re in Cereals resistance acts post-penetration the invasion sites will be open to many soil borne fungi Fungal associations may cause greater damage to the plant than either pathogen alone. 8.1.2 Materials and Methods Seed of the three wheats, Machete, GS50A and AUS4930 was sterilised, germinated and selected as in Section 3.5. Sand collected from Palmer was heat treated at 65"C for 45 minutes and allowed to cool. It was mixed thoroughly and sieved. One seed of each cultivar with 3 seminal roots of 3cm length was grown in electrical conduit tubes of 2 sizes, small (2.7cm wide by r2.5cmhigh) and large (3.7cm wide by r2.5 cm high). p. thornei was extracted from carrot cultures. One week after planting, using a truncated pipette, lml of an inoculum containing 400 P. thornei was added to each seedling. Plants were placed in a controlled temperature growth room at 2}"C,with a 12 hour day and night provided by fluorescent light tubes (65p Einstein's). There were seven replicates of each variety and tube size for each harvest time. The small tubes were harvested at2,6 and 9 days post inoculation and large tubes only at the last harvest of9 days. Nematodes were extracted using the 3 day mister extraction technique (Section 3.2.2) and counted (Section 3.3). 8.1.3 Results Data was analysed as a RCBD with two analyses conducted. Data for both analyses was converted to percent P. thornei penetration of the initial nematode inoculum density. The first ANOVA (Table 8.1) gave a comparison of the differences in variety and harvest for the small tubes, and the second (Table 8.2) acomparison between small and large tubes at the 3rd harvest time. l5l Chapter 8 Resistance Resoonse in Cereals No significant variety effect was found (Table 8.1, 8.2), but in small tubes there was a significant increase in penetration between day 2 and 6 post inoculation (Fig. 8.1). The large tubes had significantly less nematode penetration by day 9 than the small tubes (Fig.8.2). Fig 8_.! : The effect of harvest time in relation to P. thornei penetration on wheat grown at20"C in a controlled growth room in small tubes. 30 sED=3.8 -25 E Ëzo C) o .l(t)t rs t-tel ol ql 10 oil as5 0 2 6 9 Time after inoculation (days) Fig. 8.2 :Jhe_effect of tube size on the penetration of P. thornei after 9 days on wheat grown at2O"C in a controlled growth room. 30 sED=4.3 o-25 !.20(Ë c.) o .-,ot l5 él trt ôl sl 10 dl os5 0 small large Tube size t52 Chapter I Resistance Response in Cereals Table 8.1 : ANOVA of P. thornei penetration in small tubes over the 3 harvest times. d.f. m.s. v.r. hob variety ) 230.9 L53 0.225 harvest 2 1513.2 10.05 <0.001 variety.harvest 4 176.9 l.t7 o.332 Residual s3( r) 150.5 Total 61fi Table 8.2 : ANOVA of P. thornei penetration after nine days in both small and large tubes. d.f. m.s. v.r Prob. variety 2 417.5 2.17 0.129 harvest I 1566.5 8.15 0.007 variety.harvest 2 t92.9 1.00 0.337 Residual 36 192.2 Total 4t 8.1.4 Discussion As the percentage penetration did not differ significantly between varieties, the mechanism involved in P. thorn¿i resistance in the two wheat cultivars AUS4930 and GS50A act in some manner after invasion of the nematode. There are some cases where juveniles fail to penetrate resistant plants or do so in reduced numbers, but most documented resistance operates after invasion and results in the juveniles being unable to complete their development and reproduce (Howard and cotten, rgTg). The use of large and small polyethylene conduit tubes confirms (see Section 5.3) that the smaller the volume of soil medium the greater the penetration of the nematodes. The implications for the applicability of the post penetration mode of resistance in the field should be carefully considered. Invasion by the nematode, whether or not followed by a later resistance defence mechanism, may influence susceptibility to soil borne pathogens, whether they be primary or secondary invaders. A better understanding of the histopathology and migratory habits of P. thornøi throughout the 153 Chapter 8 Resistance Response in Cereals duration of the life cycle on both resistant and susceptible hosts is needed, if the chance of disease complexes involving nematodes and fungi are to be assessed. 8.2 Inheritance of P. thornei res¡stânce in the wheat cultivar, AUS4930 8.2.1 Introduction The inheritance of plant resistance to P thornei may be simple, due to a single gene (dominant or recessive), or complex, controlled by many genes. Heritable resistance to nematodes has been incorporated into many important crops, including cereals, forages, vegetables, fruits, ornamentals, tobacco, cotton and soybeans (Bingefors, 1982). There are three main sources of resistance; wild plant species, induced mutants usually produced by inadiation and plant regeneration from organs, tissues or cells producing somaclonal variants (Fassuliotis, 1987). Since plant parasitic nematodes are soilborne, the spread of new virulence genes should be relatively slow in comparison with airborne pathogens, and therefore resistant varieties should remain effective for relatively long periods (Cook, 1974). Although much of the research for sources of resistance has concentrated on nematodes with a highly specialised relationship with the host, thirty two crops have been screened for resistance to various species of Pratylenchus (Townshend, 1990). With regard to Pratylenchøs species in cereals, resistance of maize both to P. zeae and P. brachyurus is due to two dominant genes with an additive effect (Sawazaki et aI., I99I). The P. thornei resistance in wheat selection GS50A from Queensland is thought to be controlled by a single dominant gene (J. Thompson, p€Ís. comm.). There is a complex mode of inheritance for P. penetrans resistance in Medicago sativø L. (Christie and Townshend, 1992). t54 Chapter I Resistance Response in Cereals In a breeding program where a promising wheat line has been identified for resistance to an economic pathogen, the inheritance of resistance is usually investigated by comparing the F2 segregation ratios observed from crosses between the susceptible and resistant parents against hypothesised ratios. In this way many monogenic, dominant modes of inheritance have been verified, which have adhered to Flor's gene for gene hypothesis (Flor, 1956). An attempt was made to investigate the genetics of P. thornei resistance of AUS4930, since it was identified as resistant both in the laboratory (Section 5.2) and the field (Ch 7). This variety was also chosen as it carries a single gene for H. avenae resistance (F. Green, pers. comm.), in addition to P. thornei resistance. 8.2.2 Materials and Methods Glasshouse crosses were made using the susceptible wheat cultivar Schomburgk as the parent in combination with the resistant wheat 4US4930. Seed of the homozygous parent Schomburgk was obtained from the'Waite Agricultural Research Institute wheat 'Winter breeder, Dr A. Rathjen, and of AUS4930 from the Australian Cereals Collection. The Fl plants were grown for seed propagation and allowed to self fertilise and the F2 seeds were collected. All plants were grown in 8 inch pots with a nutrient rich soil mix allowing for maximum tillering capacity. Careful synchronisation of flowering times was sought because AUS4930 was an unadapted landrace originally from Iraq while Schomburgk was a locally adapted spring wheat. This synchronisation was achieved by continued weekly plantings of AUS4930 and Schomburgk over three months in Spring (Sept.- Nov.). Crossing was usually attempted mid-morning as the pollen appeared more receptive at that time. Once crossing had taken place, the emasculated heads were bagged to avoid contamination problems with drifting pollen. 155 Chapter I Resistance Response in Cereals An inheritance of resistance assay was conducted, adapted from the technique established in Chapter 5. This involved 30 selected parental seeds of both Schomburgk and 4US4930, 30 Fl seed (Schomburgk * AUS4930) and 100 of the F2 progeny (Fl seed selfed). All seeds were sterilised, germinated and selected as in Section 3.5. Each seed with 3 seminal roots of 3cm length was grown in electrical conduit tubes (2.7cm wide x 12.5cm height). The tubes were filled with Palmer soil (Section 3.7) which had been heat treated at 65oC for 45 minutes. Seedlings were inoculated 1 week post sowing with 400 P. thornei per plant in 1ml aliquots using nematodes from carrot cultures (Nicol, 1991, Section 3.1). Plants were placed in a controlled temperature growth room (Plate 8.2) at20"C, with 12 hour day and night provided by fluorescent tubes (65p Einstein's). The tubes were individually embedded in a tray of soil, with a wire grid to support the tubes. Plants were watered with tap water whenever required, so that water was not a limiting factor. Plants were harvested after 2 months and nematodes were extracted using the 3-day mister technique (Section 3.2.2) and then counted (Section 3.3.). In addition, dry weight of roots of plants was determined by placing individual plants in alfoil trays in a drying oven at 80"C for 7 days. 8.2.3 Results The results (number of nematodes per plant and root dry weight per plant (g)) were analysed as a CRD and summarised in Table 8.3. The Fl , F2 and parents were significantly different from each other (Fig. 8.3) with the resistant parent AUS4930 having significantly less nematodes than the susceptible parent. The Fl cross was almost significantly different from AUS4930 but not from Schomburgk. Further ANOVA (Table 8.3), comparing individual combinations (Schomburgk and 4US4930, 156 Chapter 8 Resistance Response in Cereals AUS4930 and F1, Schomburgk and Fl) confirmed that Schomburgk had significantly more P. thornei in its roots than the resistant parent AUS4930 (Fig. S.3). There was almost significantly less P. thornei per AUS4930 plant compared with the F1 cross. However, when the susceptible parent was compared against the Fl there was no significant difference between numbers of nematodes in their root systems. This information suggests that resistance with AUS4930 is possibly homozygous recessively inherited, which means it will only occur when the genotype is homozygous. In addition to analysing the number of nematodes per plant, the dry weight of roots(g) was also considered as a response variable when comparisons were made between individual combinations of the parents and Fl populations (Table 8.3). This revealed that the dry weight of roots did not significantly differ between the parents or Fl population. For this reason, dry weight of roots was not used as a covariate. Table 8.3 : ANOVA comparing the number of P. thornei and dry weight of roots of the parental lines, Fl and F2 populations. (The first ANOVA compares the parénts, Fl and F2 while the next three are individual comparisons) Variable : no. P. thornei per plant Variable : Dry weight roots per plant (e) d.f. m.s. v.r. Prob. d.f. m.s. v.r. Prob. Parents, Fl, F2 vanety 3 3.688+06 2.63 0.05 J .0008542 1.28 0.281 Residual 165(2 l) 1.40E+06 r74(t2) .006648 Total l 68(2 l) 177(r2) Schomburgk and AUS4930 vanety I 3404495 4.39 0.04 I .oo47t7 1.05 0.31I Residual 53(5) 774890 s2(6) .004s69 Total 54(s) s3(6) AUS4930 and Fl vanety I 2849800 3.65 0.06 I .017874 3.07 0.09 Residual s3(s) 78001 l 53(5) .005823 Total 54(s) s4(5) Schomburgk and Fl vanety I 24580 0.02 0.882 I .004223 0.72 0.40 Residual 50(8) r101264 49(9) .005863 Total 5l(8) 50(9) Further investigations of the F2 data using the Chi-square test (X2), were initiated to determine the possible number of genes involved in controlling the P. thornei resistance 157 Chapter 8 Resistance Response in Cereals in 4US4930. In order to separate the F2 data into resistant or susceptible, a cut off point for resistance is required to distinguish resistant F2 progeny from susceptible F2 progeny. The value of 1216 P. thornei per plant was selected for this purpose, being the mid-value between the mean values for the resistant and susceptible parents tested. Because the distribution of the numbers of P. thornei per plant was not distinctly separated between the two parents but showed considerable overlap (Fig. 8.4), the classification of the F2 progenies into parental type was dependent on the threshold chosen. That is, the use of 1216 P. thornei per plant to distinguish resistance from susceptibility appears sensible but it must be appreciated that the frequencies obtained are highly dependent on the choice of the threshold. The information presented suggests the AUS4930 resistance to P. thornei is controlled by a recessive allele. In order to test this the expected segregation ratios in the F2 were investigated for a single recessive locus and two independent recessive loci. fi8. l.f : Multiplication of P. thornei_on susceptible, resistant, Fl and F2 progeny wheat plants grown for 2 months at 20oC in a contiolled growth room. (a: no significant difference; b: significantly different from a; a': almosr (P<0.05) significantly different from b but not from a.) a 1800 b a' a 1600 SED = 306 Ë d 'õrë EI rlol ;l 200 0 AUS493O FI F2 158 Chapter 8 Resistance Resoonse in Cereals Fig. 8.4 : Frequency distributions of the number P. thornei per plant of parents and F1 progenies from plants grown for 2 months in a controlled growth room. 12 Schomburgk 10 I E AUS4930 I ! F,I o Ê o o (! 4 2 0 c P. thomei / plant Kesrstance rs controlled by a srngle recessrve loci. As a result a 3 (resistant) ratio would be expected in the F2 proeenv Parents AA (susceptible) X aa (resistant) F1 Aa (susceptible) F2 progeny AA, aA, Aa, aa Expected F2 ratio 3 (susceptible) : 1 (resisrant) Expected F2 plants 66 (susceptible) : 22 (resistant) Observed F2 plants 46 (susceptible) : 42 (resistant) X2 =24.24 >X2 tables (3.84) Conclusion : significant departure from Hypothesis I 159 Chapter I Resistance ResDonse in Cereals Hypothesls Z : Keslstance ls controlled recessrvely by two independent loci .{sa rÊsult a 9 (susceptible) : 7 (resistant) ratio would be expected in the F2 progeny lThis resistance can be if either of the 2 independent loci sre present in the recessive form). Parents AAA'A' (susceptible) X aaarar (resistant) F1 AaA'a' (susceptible) F2 progeny Fl gametes AA' Aa' aA' aat AA' AAA'A' AAa'A' aAA'A' aAa'A' Fl gametes Aa' AAA'a' AAa'a' aAA'a' aAatat aA' AaA'A' Aaa'A' aaA'A' aaa'A' aal AaA'a' Aaatat aaA'a' aaatat Expected F2 ratio 9 (susceptible) : 7 (resistant) Expected F2 plants 49 (susceptible) : 39 (resistant) Observed F2 plants 46 (susceptible) : 42 (resistant) X2 = 0.57 < X2 tables (3.84) Conclusion : no significant departure from Hypothesis 2. Thus it would appear there are two independent recessive loci in AUS4930 either of which results in resistance to P. thornei in the homozygous form. 8.2.4 Discussion Cook (1914) noted that the usefulness of plant resistance to nematodes is highly dependent on the genetic mechanism involved. From the statistical analysis, the resistance found in AUS4930 to P. thornei appears to be controlled by two independent recessive genes either of which results in resistance when present in the homozygous resistant form. The presence of two independent loci, either producing resistance in the one genotype, would seem unlikely from the evolutionary point of view, but not impossible. However, it should be remembered that Schomburgk 160 Chapter I Resistance Response in Cereals (susceptible parent) and AUS4930 (resistant parent) did not divide distinctly into two types. In theory, if both resistant and susceptible genotypes were pooled we should find support for a 1:1 ratio. However, as demonstrated in Fig. 8.5, the numbers of P. thornei counted in individual root systems suggested that some of the Schomburgk plants were resistant and similarly some of the AUS4930 were highly susceptible (i.e. the seed used was variable). The overlapping distribution of parents equally could be expected to affect the F2 progeny. Because of this, using 1216 P. thornei per plant as the cut off point to determine the number of resistant and number of susceptible F2 plants may be misleading and explain why significant departure from a single gene hypothesis is seen with the F2 populations examined. There are two major possible explanations for such an overlap of parental genotypes. The assay technique used may have given false results. Although 400 P. thornei werc inoculated to each plant post germination, negligible penetration occurred with some replicates, and hence there was minimal multiplication when harvested two months later. The data from Chapter 5 showed much less variation between replicates where only 10 replicates were used, but in this experiment 30 parents and Fl and 100 F2 were assessed. Christie and Townshend (1992) found their assay technique for counting the numbers of root lesion nematode also produced rather variable results with large standard errors. They pointed out that for a breeding program to be successful techniques which would give more precise results would be essential. Nelson et ¿/. (1985) suggested using root weight as an indicator of resistance rather than nematode numbers for measuring resistance of Pratylenchus spp. to the genus Prunus. However, this approach was not supported here with P. thornei and wheat as the root dry weight did not significantly differ between varieties. 161 Chapter I Resistance Response in Cereals The second possibility is that the original parental seed was not "true to type" for P. thornei resistance, given that crossing was performed with several plants of both parents. This hypothesis is quite plausible for AUS4930 as the seed (although multiplied by the Australian'Winter Cereals Collection) was originally a landrace from Iraq and the genetic diversity is expected to be quite high. Results from field sampling of the resistant AUS4930 (Ch 7) late in the growing season showed some plants with high nematode populations comparable to the susceptible wheat Warigal. In addition, the heads showed morphological differences, with and without awns and with colours varying between white and dark cream, further supporting lack of genetic uniformity. It is also possible that Schomburgk, although homozygous for the disease and quality traits it was purposely breed for, may carry some heterozygosity within loci for P. thornei resistance. A possible way to test such a hypothesis could be to obtain several single heads of both parents and re-run the assay. If seed from some individual heads produced highly variable results while others were uniform this would suggest variation in the parental type. If there was a similar high variability in seed from all heads this would implicate problems with the assay technique. It is important to note that if the F2 segregation had been more reliable, the confirmation of the F2 ratios could have been verified by analysis of the F3 families. As a result segregation for resistance and susceptibility would have been enhanced providing a more precise confirmation of the numbers of genes involved in AUS4930 resistance. If some reselection of both resistant and susceptible parents were carried out and the above experiment was repeated with reliable parental separation the investigation of the F3 populations would be highly desirable. Although the attempt to determine the number of genes involved in AUS4930 resistance to P. thorn¿i was not conclusive, the finding of the apparently recessive t62 Chapter I Resistance Response in Cereals nature of resistance and that the mechanism of P. thorn¿i resistance in both GS50A and AUS4930 acted after penetration should be carefully considered. Nematode invasion is likely to predispose the plant to infection by other soil borne pathogens (Lawn and Sayre, 1992). As a consequence, the mechanism of resistance has implications for the exploitation by soil fungi. It is possible that similar numbers of P. thornei invade the cortex of resistant and susceptible plants throughout the life of the plant, and the number of entry sites may not differ, but the corresponding multiplication may. An understanding of the migratory and histopathological behaviour of P. thornei is of paramount importance, particularly for South Australia which has numerous soil borne fungi (many considered to be secondary invaders). Current evidence suggests that p. thornei is a non-random invader, attracted to pre-invasion sites (Baxter and Blake, 1967). The reasons for this behaviour are poorly understood. It is obvious from the literature and the above work that little information exists with regard to understanding the mechanisms and genetic control of P. thornei resistance in wheat. Further selection to improve the purity of P. thorn¿i resistance of GS50A and AUS4930 is essential. 163 Chapter 9 N ematode/ F un p al I nt e rac tiotts Chapter 9 Nematode/Tungal Interactions 9.1 Introduction Under aseptic laboratory conditions, P. thornel alone can cause damage to cereals (Nicol, 1991, Ch. 6). In the field plants are rarely ever subject to the influence of only one potential pathogen. This is especially true of soil borne pathogens (Powell, l97I). In field situations world-wide, P. thornei is also known to contribute to yield reductions of cereals (Section 2.8). The first report of a nematode-fungus interaction was made by Atkinson in 1892 where increased disease severity was found with Fusarium wllt on cotton grown in soil coinfected with Meloidogyne sp. Species of Pratylenchus appear to be the dominant form of nematode involved in interactions with Verticillium wilt fungus (Powell, I97I). Usually either pathogen is capable of causing disease but damage is much greater when both are present together. In addition to 'wilt' complexes with Pratylenchus, there are numerous reports of Pratylenchus and associated complexes with 'soil-borne' fungi (Section 2.5). Early work was conducted on root rot of winter wheat in Canada, where P. neglect .t was found to interact with Rhizoctonia solani resulting in significant reductions in wheat growth (Mountain, 1954; Benedict and Mountain, 1956). Since this initial work, numerous soil borne fungal associations with Pratylenchus species have been documented (Powell, 197 l). In South Australia, soil borne fungi are numerous and commonly present in cereal growing regions (4. Rathjen, pers. comm.). Taheri et al. (1994) found that in South Australia wheat roots and the nematode P. neglectus were commonly associated with Fusarium oxysporum, F. acuminatuffi, F. equiseti, Microdochium bolleyi, Gaemannomyces graminis, Bipolaris sorokiniana, Pythium irregulare, Pyrenochaeta t64 Chapter 9 N emato de/ F un gal I nt e rac tio ns terrestris, Phoma sp. and Ulocladium atrum. As previously discussed (Ch. 4), the two Pratylenchur species, P. neglectu.r and P. thornei are commonly found in southern Australia and frequently together. Preliminary evidence suggests fungal associations with P. neglectus are responsible for increased damage to wheat (4. Taheri, pers. comm.). Due to the frequency of soil fungi in South Australia and similar widespread distribution of Pratylenchus, preliminary investigations were undertaken to investigate any interaction of two soil borne fungi, Microdochium bolleyi and F. acuminatum with the two nematode species, P. thornei andP. neglectus. These fungi were selected as they are two of the most common soil borne fungi in South Australia (4. Taheri, pers. comm.), and are both considered to be secondary pathogens. This work was undertaken in collaboration with Mr. Abdolhossein Taheri, Department of Plant Science, Waite Campus. 9.2 Materials and Methods Machete seeds were sterilised, germinated and selected as in Section 3.5. Sandy soil (Ch. 3.7) from wheat fields near Roseworthy Campus of the University of Adelaide was steam pasteurised at 7O"C for 40 minutes, then aerate d for 72 hours and sieved through a2mm sieve. Fungal inoculum of M. bolleyi and F. acuminatum was prepared on millet seed that had been placed in plastic autoclave bags and autoclaved at I20"C for one hour on each of three consecutive days. The fungal inoculum from PDA medium was added to each plastic autoclave bag containing sterilised millet seed (0.5kg), grown for four weeks at 25"C and then air dried in a laminar flow cabinet for one week prior to use. 300rnl plastic cups (as in Plate 6.2), with no drainage, were filled wirh 42Og air dried soil. 165 Chapter 9 N emat ode/ F unp a I I nt e rac t io ns Fungal inoculum of F. acuminatum or M. bolleyi was added to the soil at IVo wlw. One layer of fungus was added after 1509 of soil was in the cup and a second another 1509 soil. The remaining 1209 of soil was added to fill the cup. Pregerminated Machete seeds were sown in each cup at a depth of 1.5cm (1 seed per cup). P. thornei and P. neglectus were extracted from carrot cultures as described in Section 3.1. The nematodes were added in a volume of lml using a truncated pipette, at the densities of 0, 2000, 6000 and 12000 per plant, as close to the seedling as possible. Sterile distilled water was added for the 0 nematode treatment. The experiment was set up as a split plot with 6 replicates. There were 2 harvest times (7 and 10 weeks post inoculation) were randomised to the two whole plots within each of the six replicates (blocks). Two nematode types (P. neglectus and P. thornei) at 4 different initial densities, and two fungi (M. bolleyi and F. acuminatum) were randomised to the 24 subplots within each whole plot. Plants were grown in a controlled temperature room at 23"C with a 12 hour day length and light intensity of 65pEinsteins. This temperature was selected as it is optimal for both fungal and nematode species. Plants were harvested 7 and 10 weeks post inoculation. The soil was gently washed from the root system. Nematodes were extracted over a period of 4 days using mister extraction (Section 3.1), was counted (Section 3.3). At each harvest time, the root lesions were scored from 0-5 (O=healthy roots and 5=complete lesioning of whole root system) and the number of tillers per plant (excluding main tiller) were counted. Dry weights of shoots and roots were recorded after drying at 80oC for 7 days. A 2cm root segment from each treatment was sampled and fixed in FAA preservative for staining nematodes and fungus (Section 3.4.2). 166 Chapter 9 N emat ode/ F un p al I nt e rac t io n s 9.3 Results The data (no. nematodes/plant; multiplication rate (no. nematodes per plant/ initial nematode density); dry weight roots (g); dry weight shoots (g); total dry weight (g); root lesioning/plant and number tillers/plant) were analysed as a SPA. Where the original analysis showed heterogeneity of variance the data was either log transformed, logs(x+1), or square root transformed (sqrt x+0.5). There are two significant 3-way interactions for the square root transformation of the number of nematodes per plant. The first of these is a 3-way interaction between harvest time, nematode species and initial nematode density. In general, as the initial nematode density increases the number of nematodes per plant also increases, however at harvest 2, P. thorn¿i shows little increase at the highest density whereas P. neglectus exhibits much higher numbers at the highest density (Fig. 9.1). The second is a 3-way interaction between harvest time, nematode and fungal species. Plants with F. acuminatumhad greater number of both P. thornei and P. neglectus at harvest 1, but by harvest 2 there was an increase in the number of P. thornei with or without fungi but no difference between treatments. Similarly the number of P. neglectus increased, but the highest number of nematodes was in the absence of fungi (Fig.9.Z). A 3-way interaction between harvest, nematode species and fungus for the multiplication rate of nematodes per plant is illustrated in Fig. 9.3. This has the same pattern as the numbers of nematodes per plant. From the analysis we also note that as initial density increases the multiplication rate of the nematode decreases (Fig. 9.4). There are two significant 2-way interactions for shoot dry weight per plant(g). First nematode density had little effect at harvest 1 but much greater effect at harvest 2 (Fig. 9.5). The significant Z-way interaction between harvest time and fungus showed both 167 Chapter 9 N ematode/F un p al I nt e rac tions fungi increased the shoot dry weight however the increase at harvest 2 was much greater than at harvest 1 (Fig. 9.6, Plate 9.1 and 9.2). Table 9.1 : ANOVA variables examining the effect of fungus, nematode density and species over two harvest times on the wheat cultivar Machete. d.f. m.s. v.r Prob. Variable : Square root (No. nematodes/plant + l) block stratum 5 4958 block.wolot stratum harvest I t07248 19.20 <0.007 Residual 5 5587 block.wplot. subplot stratum nemtype 1 8054 3,01 0.08s fungus 2 341 0. l3 0.880 nemden 152367 56.90 <0.001 harvest.nemtype I t274 0.48 0.491 harvest.fungus 2 18273 6.82 0.001 nemtype.fungus 2 2587 0.97 0.383 harvest.nemden 4495 1.68 0.190 nemtype.nemden 742t 2.77 0.06s fungus.nemden 6 r49t 0.s6 0.694 harvest. nemtype.fungus 2 9524 3.56 0.031 Fig.9 .2 harvest.nemtype.nemden J 9878 3.69 0.027 Fig.9 .1 harvest.fungus.nemden 6 3708 1.38 0.24r nemtype.fungus.nemden 6 tt79 0.44 0.779 Residual l68(6) 2678 Total 277/10\ Va¡iable : Multiplication rate of nematodes/plant block stratum 5 9.536 block.wplot stratum harvest I 362.824 6l.06 <0.001 Residual 5 5.942 block.wplot. subplot stratum nemtype I 14.338 1.53 0.218 fungus 2 0.164 0.02 0.983 nemden 3 28.257 3.02 0.052 Fig 9.4 harvest.nemtype I 2.755 0.29 0.588 harvest.fungus 2 40.209 4.29 0.015 nemtype.fungus 2 10.006 1.07 0.346 harvest.nemden J 3.034 0.32 0.724 nemtype.nemden J 9.625 1.03 0.360 fungus.nemden 6 7.939 0.8s o.497 harvest.nemtype.fu ngus 2 30.784 3.29 0.040 Fig.9.3 harvest.nemtype. nemden 3 14.890 1.59 0.207 harvest.fungus.nemden 6 5.129 0.55 0.701 nemtype fungus.nemden 6 4.434 0.47 0.755 Residual I 68(6) 9.364 Total 209/6'\ 168 Chapter 9 Interactions d-f. m.s. v.r, Prob. Va¡iable: Þry weight shootVplant (g) block stratum 5 0.16079 block.wolot stratum harvest 1 t6,72968 309.24 <0.001 Residual 5 0.05410 block.wplot. subplot stratum nemtype 1 0,05338 3.58 0.060 fungus 2 3.46281 232.26 <0.001 nemden J 0.t3482 9.04 <0.001 harvest.nemtype I 0.01371 0.92 0.339 harvest.fungus 2 0.13916 9.33 <0.001 Fig.9.6 nemtype.fungus 2 0.00091 0.06 0.94t harvest.nemden 3 0.04765 3.20 0.024 Fig.9.5 nemtype.nemden J 0.01257 0.84 0.472 fungus.nemden 6 0.02007 l.35 0.237 harvest. nemtype. fungus 2 o.01426 0.96 0.386 harvest.nemtype.nemden J 0.00603 0.40 0.750 harvest.fungus.nemden 6 0.01082 0.73 0.629 nemtype.fungus.nemden 6 0.01657 l.1l 0.356 Residual 232(4) 0.01491 Total 283ø\ Variable : Dry weight roots/plant (g) block stratum 5 0.39057 block.wolot stratum ha¡vest I 0.85594 5.00 0.076 Residual 5 o.t1L2t block. wplot. subplot stratum nemtype I 0.38964 5.04 0.026 fungus 2 1.27373 16.47 <0,001 nemden 3 0.57571 7.44 <0.001 harvest.nemtype t 0.35423 4.58 0.033 Fig. 9.8 harvest.fungus 2 l.t5tt6 14.88 <0.001 nemtype.fungus 2 0.35648 4.61 0.011 harvest.nemden 3 0.10483 1.36 0.257 nemtype.nemden 3 0.03355 0.43 0.729 fungus.nemden 6 0.44199 5.7r <0.001 harvest. nemtype. fun gus 2 0.15205 r.97 0.142 harvest.nemtype.nemden 3 0.11318 1.46 0.225 harvest.fungus.nemden 6 0.33807 4.37 <0.001 Fig.9.7 nemtype,fungus.nemden 6 0.062t1 0.80 0.569 Residual 221(rs) 0.07734 Total 272rr5), 169 Chapter 9 N ematode/ F un g al Inte raction s d.f. m.s. v.r Prob. Va¡iable : Toøl dry weighlplant (g) block stratum 5 0.9900 block.wplot stratum harvest I 9.8572 27.43 0.003 Residual 5 0.3593 block. wplot.subplot stratum nemtype I 0,1791 t.57 0.2r2 fungus 2 5.9711 52.32 <0.001 nemden 3 1.3248 11.61 <0.001 harvest.nemtype t 0.5932 5.20 0.024 Fig.9.11 harvest.fungus 2 1.2051 10.56 <0.001 nemtype.fungus 2 0.3859 3.38 0.036 Fig.9.10 harvest.nemden 3 0.0870 0.76 0.516 nemtype.nemden 3 0.02t9 0.19 0.902 fungus.nemden 6 0.6785 5.95 <0.001 harvest.nemtype.fu ngus 2 0.2720 2.38 0.095 harvest. nemtype. nemden 3 0,1401 t.23 0.300 harvest.fungus.nemden 6 0.3506 3.07 0.007 Fig.9.9 nemtype.fungus.nemden 6 0.1231 1.08 0.376 Residual 218(18) 0.1 l4l Total 269fi8\ Variable : Root lesioning/plant block stratum 5 0.4421 block.wolot stratum harvest I 14.8640 333.97 <0.001 Residual 5 0.0445 block.wolot.subolot stratum nemtype I 0.2770 1.24 o.267 fungus 2 9.0t79 40.27 <0.001 nemden J 53.5480 239.ts <0.001 harvest.nemtype I 0.23s3 1.05 0.306 harvest.fungus 2 r.9730 8.81 <0.001 nemtype.fungus t 0.1255 0.56 0.572 harvest.nemden J 1.2496 5.58 0.001 Fis.9.t4 nemtype.nemden J 0.1131 0.51 0.679 fungus.nemden 6 0.7717 3.45 0.003 Fig.9.l3 harvest.nemtype.fun gus 2 1.2498 5.58 0.004 Fig.9.l2 harvest.nemtype.nemden 3 0.4590 2.05 0.108 harvest.fungus.nemden 6 0.3789 r.69 0.124 nemtype. fungus.nemden 6 o.t76t 0.79 0.581 Residual 226(10) 0.2239 Total 277(tO) 170 Chapter 9 N e matode/ F un gal I nte rac t io ns d.f. m.s. v,r. Prob. Variable : No. tillers/plant block stratum 5 8.4661 block.wplot stratum harvest I 2.0387 2.62 0.t67 Residual 5 0.7792 block. wplot,subplot stratum nemtype I 0.0772 0.14 0.711 fungus 2 62.6843 l l1.63 0.001 Fig.9.15 nemden J 0.8534 r.52 0.2t0 harvest.nemtype I 0.3516 0,63 0.430 harvest.fungus 2 0.9928 r.77 0.173 nemtype.fungus 2 1.5705 2.80 0.063 harvest.nemden J 0.2344 0.42 0.741 nemtype.nemden J 0.5900 1.05 0.371 fungus.nemden 6 0.8815 1.57 0.r57 harvest. nemtype. fungus 2 0.6053 1.08 0.342 harvest.nemtype.nemden 0.3339 0.59 0.619 harvest.fungus.nemden 6 0.3202 0,57 0.754 nemtype.fungus.nemden 6 0.3 188 0.57 0.756 Residual 228(8) 0.5615 Total 279(8\ The 3-way interaction between harvest time, fungus and initial nematode density on the dry weight of root per plant (g) is illustrated in Fig,9.7 . There was a general trend at all harvest times and fungal treatments for a decrease in root weight with increasing initial nematode density. Unlike shoot dry weight, the dry weight of root systems between harvest times did not change except that M. bolleyi caused more root growth than was found in the control or with F. acuminatumat harvest 1. The two-way interaction between nematode species and harvest time revealed that P. neglectus significantly reduced the root dry weight of Machete at harvest l, while both P. neglectus andP. thornei reduced root dry weight at harvest 2 (Fig 9.8). The root dry weight per plant was less at harvest 2 than harvest 1, unlike the shoot dry weight per plant. t7t Chapter 9 N e matode/F unp al Int eractions Fig. 9.L : Effect of interaction between harvest, nematode species and initial density on the number of nematodes extracted from Machete wheat root systems. 2û 2û 220 € ! 2000 nematodes/plant Þ. 2æ E 6000nematodes/plant ú 180 I l2ooonemarodes/planr U) 160 SED = t8.ZS o I 1,10 Ê 120 Ec) 100 I(! o 80 .:::.: z :,.::,: 60 40 '.;.:.t. 20 ..:.::..:: 0 Harvest I Ha¡vest 2 Harvest I Ha¡vest2 P. thomei P. neglectus Fig. 9.2 :_Effect of interaction between harvest, nematode and fungal species on the number of nematodes extracted from Machete wheat root systems. 2A0 175 I Control (€ o. ø M. bolleyi ú 150 I F. acuminatum SED = 18.75 U) r 125 0) 100 o ô CË 75 tr z(.) 50 25 0 Harvest I Hawest 2 0 Harvest I Harvest 2 P. thornei P. neglectus 172 Chapter 9 N emato de/F un pal I nt e ractio ns Fig. 9.3 : Effect of interaction between harvest, nematode and fungal species on the nematode multiplication rate per Machete wheat root system. 8 7 0) I control G M. bolleyi & 6 P É I F. acuminatum (t sED 0.99 o 5 = o À 4 r5o lj 3 z6) 2 0 Harvest I Hawest 2 0 Harvest I Harvest 2 P. thomei P. neglectus Iig.g.¿ : Effect of initial nematode density on the nematode multiplication rate per Machete wheat root system. 4.50 SED = 0.5 4.25 c) úG 4.00 '.5 d o o. 3.75 ¿ q) E d 3.50 zc) 3.25 3.00 2000 6000 1 2000 Initial Nematode Density 173 Chapter 9 N ematode/F un pal I nte ract io ns Fig. 9.5 : Effect of harvest time and initial nematode density on the shoot dry weight of Machete. 1.50 I O nematodes/plant E ZOOO nematodes/plant 1.25 I OOOO nematodes/plant èo ø 12ooo nematodes/ptant rú SED = 0.02 o 1.00 èo q) È o.75 : U) 0.50 o.25 0.00 Harvest 1 Harvest 2 Fig. 9.6 : Effect of harvest time and fungal species on the shoot dry weight of Machete 1.75 I control 1.50 $ u. boueyi bo 2 I F.acuminatum t.25 CÉ SED = 0.03 = è0 o 1.00 'L ! 0.75 o rt) 0.50 0.25 0.00 Harvest I Harvest 2 t74 Chapter 9 N e mato de/ F un g al I nt e ra c t io n s Fig. 9.7 : Effect of interaction between harvest, nematode species and initial nematode density on the root dry weight per Machete wheat root system. t.2 1.1 | 0 nematodes/plant 1.0 E zooo nematodes/ptant öo oooo nematodes/plant Y o.e I d ø 12OOO nematodes/ptant E 0.8 SED = 9.12 0.7 .Eo() :..ì È :: È- 0.ó :: 0.s ëo ú o.4 ir 0.3 i:: ':.. o.2 0.1 I 0.0 Hüvest I Hwest 2 Hdvest I Hæesr 2 Hüvest I Hflest 2 Control M. bolleyi F. acuminatum Fig. 9.8 : Effect of interaction between harvest and nematode species on the root dry weight per Machete wheat root system. 0.9 0.8 I Control o.7 4 P.thornei €9 I P. neglectus 0.6 SED 0.09 So. = .Eo 0.5 ìq) 0.4 !¿' & 0.3 0.2 0.1 0.0 Harvest I Harvest 2 175 Chapter 9 N ematode/F unp al I nt e ractions Fig. 9.9 : Effect of interaction between harvest, nematode species and initial nematode density on the total dry weight per Machete wheat root system. 2.75 I OnematodeVplant 2.50 f 200OnematodeVplant 2.25 ! 600Onematodes/plant èo :.: @ l200OnematodeVplant 2.00 ..:. d SED = 0.15 o- .:.. r.75 i: .:. .:, ':., bo i:: () 1.50 :::l ..:. È ::.1 ::.:: i ,' t.25 ':.. 1.00 F l.'. i :... 0.75 :.1 :. :1.: 0.50 :. :i :: t,,' o.25 .: i',, :: 0.00 Conkol M. bollei F. acxninofrtnt Contol M. bolleyi F, acuntinsilut Harvest I Harvest 2 Fig. 9.10 : Effect of interaction between nematode and fungal species on the total dry weight per Machete wheat root system. 2.50 2.25 I Control 2.N à M. bolleyi ôo I F.acuminatum (! 1.75 SED = 0,07 1.50 ìòo ì r.25 1.00 cd o F 0.75 0.50 0.25 0.00 P. thornei P. neglectus 176 Chapter 9 N ematode/F un p al I nt e ractions Fig. 9.11 : Effect of interaction between time of harvest and nematode species on the total dry weight per Machete wheat root system. 2.5 I P. thornei 20 S P. neglectus òo SED = 0.08 rd Þ. 1.5 èo c) 'o'Þ 1.0 (Ë F 0.5 0.0 Hawest I Harvest 2 Fig. 9.12 : Effect of interaction between harvest, nematode and fungal species on the degree of root lesioning per Machete wheat root system. 2.5 I Control S u. bouevi 2.O I F. acumitnrum rñ SED = 0.13 .l. bo 1.5 o o o ú 1.0 0.5 0.0 Hawest I Harvest 2 Harvest I Harvest 2 P. thomei P. neglectus 177 Chapter 9 Interactions Fig. 9.13 : Effect of interaction between fungal species and nematode density on the degree of root lesioning per Machete wheat root system. 3.0 I Onematodes/plant S 20OOnematodes/plant 2.5 | 6000nematodeVplant @ l2000nematodes/plant !ñ SED 0.14 I 2.0 = bo 1.5 () o ô ú 1.0 0.5 0.0 Control M. bolleyi F. acuminatum Fig.9.14 :.Effect of interaction between harvest and nematode density on the degree of root lesioning per Machete wheat root system. 30 | 0 nematodes/plant 25 E 20o0ne-utodes/plant I 6000nemarodes/plant r) E 12000nematodevplant 9 2.o bo SED = O-I 3 rs ú 1.0 0.5 0.0 Harvest I Harvest 2 178 Chapter 9 N e mato de/F un p al I nt e ractions Fig. 9.15 : Effect of fungal inoculum on the number of tillers per Machete wheat root systems. 4.O 3.5 I Control M. bolleyi 3.0 fr I F. acuminatum F SED 0.1I 2.s = IE c E z.o c) 1.5 =F 1.0 0.5 0.0 The significant 3-way interaction between harvest time, fungus and initial nematode density for the variable total dry weight per plant (g) is illustrated in Fig. 9.9. P. thornei and P. neglectus, \/ith and without associated fungi, caused significant reductions in total dry weight per plant at high initial densities for both harvest times. The presence of M. bolleyi and F. acuminatum led to an increase in total dry weight relative to the nematode alone (Fig. 9.9), inespective of nematode species (Fig. 9.10). A signif,rcant 2-way interaction between nematode species and harvest revealed the total dry weight with both nematode species was greater at harvest 2 than harvest 1 (Fig. 9.1 1). It was also noted that nematode species responded differently at harvest 1, with P. neglectøs giving lower total dry weight than P. thornei, whereas the opposite occurred at harvest 2 (Fig.9.1 1). Chapter 9 N ematode/F un p al I nt e ractions Plate 9.1 : Representative Warigal plants from the three nematode fungus treatments; 2,000 P. thornei + M. bolleyl (left), M. bolleyi alone (middle) and control plants (right). The plants were grown for 10 weeks at 2O"C in a controlled growth room. The presence of fungi with or without nematodes caused a stimulation of shoot growth. Plate 9.2 : Representative Warigal plants from the three nematode fungus treatments; 2,000 P. thornei + F. acuminatum (left), F. acuminatum alone (middle) and control plants (right) Plants were grown for 10 weeks at2O"C in a controlled growth room. The presence of fungi with or without nematodes caused a stimulation of shoot growth. I ,t I Ltb 2000 ) L I I i conlrol t Chapter 9 N e matode/ F unp al Int e rac t ions Plate 9.3 : Representative V/arigal replicates from the three treatments, M. bolleyi (left), 12,000 P. neglectus (middle) and 12,000 P. neglectus + M. bolleyi (right). Plants were grown for 10 weeks at20"C in a controlled growth room. In the presence of nematodes with fungi the degree of root lesioning was much greater than either nematode or fungi alone as indicated by the dark cortical lesions. Plate 9.4: Representative \ù/arigal replicates from the three treatments, control with no nematodes (left), 6,000 P. thornei + F. acuminatun (middle) and 12,000 P. thornei + F. acuminatur¿ (right). Plants were grown for 10 weeks at 2O"C in a controlled growth room. In the presence of nematodes with fungi the degree of root lesioning was much greater than either nematode or fungi alone as indicated by the dark cortical lesions. Pn 12000 Pn 12000 Mb Mb Pt 12000 Fa Control Pt 6000 Fa Chapter 9 Plate 9.5 : Stained Machete root system l0 weeks after growth in 300m1 cups, initially inoculated with 12,000 P. thornei andM. bolleyi. Evidence of masses of P. thornei (dark green) and eggs (oval shaped rods) in the cortical cells of the seminal root system. Nematodes were not seen to penetrate the stele. (1cm=50¡r). Plate 9.6 : Stained Machete root system 10 weeks after growth in 300m1 cups, initially inoculated with 6,000 P. neglectøs and M. bolleyi. Evidence of extensive cortical degradation resulting in the loss of outer cortical layers. Nematodes were not seen to penetrate the stele. (lcm = 50p). .-¡ ., r¡ Chapter 9 N ematode/ F un R al Int erac ti ons Plate 9.7 : Stained Machete root system 10 weeks after growth in 300m1 cups, initially inoculated with 2,000 P. neglectøs and M. bolleyi. A cortical lesion can be seen developing by the dark green regions associated with several nematodes and masses of P. thornei eggs lying parallel to the axis of the root. (1cm=50p). Plate 9.8 : Stained Machete root system 10 weeks after growth in 300m1 cups, initially inoculated with 6,000 P. neglectus and M. bolleyi. Within both seminal and lateral roots the deposition of eggs (oval shaped rods) in rows distributed among the cortical cells was commonly seen, particularly in regions where many nematodes were present. (1cm=50p). t79 Chapter 9 Nematode/FunøaI Interactions Presence of either M. bolleyi or F. acuminatum increased root lesioning with both p. thornei and P. neglectus, with greater lesioning over time (Fig. 9.12,Plate 9.3) except F. acuminatum for P. thornei. A significant2-way interaction between initial nematode density and fungal species showed increasing initial density resulted in a corresponding increase in the severity of root lesioning except for the highest density of nematodes for the control (Fig. 9.I3, Plate 9.4). The degree of lesioning increased with time and increasing initial nematode density (Fig 9.14), however 2000 nematodes per plant were much more similar to higher densities at harvest 2. Tillering was significantly increased when the nematode was present with fungi, and more so with F. acuminatum (Fig.9.15, Plate 9.1 and9.2). 'When Machete wheat roots were stained at harvest 2, fungal spores as well as nematodes were seen inside the cortex of the root. The high initial nematode densities were associated with many nematodes usually found in groups along the cortex (plate 9.5). Cortical degradation was evident, whatever the initial nematode densities but was particularly severe at high initial densities (Plate 9.6). It was more severe when both fungi and nematodes were present, and did not occur in roots with fungi alone. Fungi appeared to be more uniformly distributed the roots than nematodes. Nematodes "vithin were found in both the seminal and lateral roots but were not present in all parts of the root. Lesion formation was cortmonly associated with nematode proliferation (plate 9.7). Adult nematodes were often seen associated with a line of eggs in the cortical tissue (Plate 9.8). 9.4 Discussion Infection by one pathogen alters the host response and subsequent infection by another pathogen (Powell, I97I). The results reported in this chapter suggest that the 180 Chapter 9 N ematode/ F un g al I nt e ract io ns interaction of M. bolleyl and F. acuminatum with the root lesion nematodes P. thornei and P. neglectus does affect the way the host (in this case Machete wheat) responds. Plant parasitic nematodes are primary plant pathogens and capable of causing important plant diseases (Prot, 1993). This is verified in this experiment, where both P. thornei and P. neglectus caused significant reductions in total dry weight at high initial densities, suggesting that both nematodes are pathogenic on wheat in their own right. All root parasitic nematodes cause mechanical injuries as they penetrate within or feed on root tissues, providing ready avenues for the entry of other pathogens (Prot, 1993). However, mechanical wounding does not always promote fungal penetration within root tissues. There is strong evidence, particularly with the sedentary nematodes such as root knot nematodes, that physiological and/or biochemical changes predispose their host to fungal pathogens (Prot, 1993). However, the precise physiological and/or biochemical changes induced or produced by the nematodes which predispose their hosts to fungal pathogens or directly enhance the invasion and development of pathogenic fungi in host tissues are not known (Prot, 1993). The inoculum density of each pathogen relative to the other was found to affect the expression of the disease complex. Because plant parasitic nematodes reproduce only in the presence of a living host, organisms such as fungi, particularly those with a similar histopathology to the nematode, are likely to be most important. Some soil fungi which are not normally parasitic on plants become parasitic on roots infected with nematodes (Powell, I97L; Powell et aI., I97I). In general, synergistic interactions, nematodes provide an opportunity for fungal pathogens to show their greater pathogenic capabilities (Hasan, 1993). l8l Chapter 9 N e matode/ F ung al I nt e ractions Both the fungi (M. bolleyi and F. acuminatum) and nematodes (P. thornei and P. neglectus) tested here have similar ecological niches. M. bolleyi has been found in cortical and epidermal cells (Spiegel and Schönbeck, 1991) and can be isolated in high frequency from cereal roots (Sprague, 1948;Murray and Gadd, 1981). It is considered to be non-pathogenic (Liljeroth and Bäath, 1989) and is largely restricted to the invasion of naturally senescing cortices of cereal grasses (Kirk and Deacon, 1987). However, it was suggested to be the primary root rot fungal coloniser on cereals in the eastern prairies of Canada (Steirz and Bernier, 1989). F. acuminatum has similar pathology to M . bolleyi, penetrating epidermal cells, but hyphae are rarely observed to enter wounds directly (Stutz et al. ,1985). Both fungi are found in high levels in late July and August in South Australian cropping regions (Vanstone, l99I), and M. bolleyi is known to be a late coloniser of barley roots (Liljeroth and Bäath, 1989). Evidence from field work suggests higher numbers and multiplication of P. thornei and, presumably P. neglectus in the latter parts of the growing season (Pattison, 1993), primarily determined by favourable environmental conditions. In this experiment, P. thornei and P. neglectus appeared to increase in number and multiplication rate in the presence of fungi after 7 weeks. This increase also occurred in egg plant and tomato infected with Verticillium and P. penetrans (Mountain, 1954). Increased reproduction of Pratylenchus occurred in the presence of Verticillium dahliae on peppermint (Faulkner and Skotland, 1965). The interaction with Verticillum could be explained by the migration of the endoparasitic nematode to fresh feeding sites, and also the growth promoting substances produced in response to fungus-host- nematode interactions (Faulkner and Skotland, 1965; Faulkner and Bolander, 1969). In the experiment described here increased nematode numbers and multiplication at low initial densities may be explained by the similar niches and histopathology of both nematodes and fungi. 182 Chapter 9 N emato de/F un p a I I nt e rac tions However, after 10 weeks of combined exposure with nematode and fungus, the number and multiplication of P. neglecrøs significantly declined relative to the control, but P. thornei numbers increased in combination with M. bolteyi. Decreased multiplication rate of the nematode in combination with the fungus relative to the nematode alone may be associated with the damage sustained to the host. The traditional concept of specific aetiology stipulates that a single agent operating under prescribed conditions is the cause of a given disease (Powell, I97I).In contrast, the doctrine of predisposition has contributed to a much better understanding of disease development (Powell, l97I), particularly in the field situation. This is of extreme importance, particularly with regard to plant resistance to parasitic nematodes and their fungal associates. Nematodes are known to break resistance to fungal infection in crop cultivars and it is logical that the contrary may occur, that fungal pathogens might also be involved in reducing resistance of cultivars to nematode infection (Hasan, 1993). This is an important consideration and warrants further investigation. The control of p. thornei and P. neglectus on cereals is currently being investigated to allow breeding of resistant cereal lines. It will be important to identify the mechanisms of resistance in order to avoid resistance break down by the interacting fungal organisms in the soil system. If the form of plant resistance precludes the nematodes from penetrating the plant, then there is little concern, but if the resistance acts after penetration (as it does with a number of sedentary nematodes (Hasan, 1993) then the breeding strategy may need to incorporate fungal resistance(s) as well. Unfortunately, little is known about varieties with resistance to either nematode or the mechanisms behind the resistance. Nematode numbers were found to increase with increasing nematode density while the multiplication rate declined. This is consistent with population changes associated with 183 Chapter 9 Interactions other migratory endoparasitic nematodes (Section 2.8). In this experiment, increases in nematode number and density increased the severity of root lesioning, with the degree of severity increasing with time. Lesioning was worse at high initial densities (6000 and 12000 nematodes per plant) with both M. bolleyi and F. acuminatum . Taheri et al. (1994) found similar increased root lesioning with the closely related species P. neglectus and with coÍtmon root rotting fungi. The measured growth parameters of Machete were all found to be significantly affected by both P. thornei and P. neglectus. In general, increasing nematode density, with or without a fungal combination, was found to decrease the growth parameters of root, shoot and the suûrmation of these. However, there was some evidence that at low initial densities (2000 nematodes per plant) plant growth was stimulated particularly up to 7 weeks. This was also reported by Nicol (1991) and in Ch. 6. However, because P. thornei is a migratory nematode and multiplication occurs continuously throughout the growing season in the presence of a host it is likely that damaging densities would override this stimulatory effect with time, as appeared to have happened by week 10. Shoot dry weight was significantly higher in plants inoculated with fungi in combination with nematodes at both harvest times. This was most strongly correlated with an increased tillering capacity of Machete. F. acuminatum did not significantly affect root dry weight, but M. bolleyi significantly stimulated root growth at the first harvest. However, by 10 weeks there was no difference between the control, F. acuminatum or M. bolleyi. Nematode species with closely related biology and feeding habits may present differences in their ability to predispose a plant to infection by the same fungus (Prot, 1993). This appears to be the case here, where P. neglectus limited root growth of 184 Chapter 9 Interactions Machete earlier than did P. thornei. It is possible that P. neglectus invaded roots earlier in greater numbers than P. thornei or that the plant cells were more damaged by P. neglectus saliva. As discussed in Chapter 6, P. neglectus left roots faster than P. thotnei, also suggesting possible differences in biology. However, by the second harvest after 10 weeks, there was no distinction between the root dry weight of plants with either P. thornei or P. neglecføs. Regions of extensive cortical degradation were commonly associated with many nematodes and eggs lying parallel to the long axis of the root nematode. This observation has been commonly made in the work of Orion er al., (1979) and Baxter and Blake (1968). Overall, the total dry weight was stimulated in the presence of fungi with or without nematodes at both harvest times. The magnitude of nematode multiplication was reduced by both fungi relative to the control over the two harvest periods of 7 and l0 weeks. It is possible that the apparent stimulation of growth was an initial response to damage, namely an attempt by the plant to compensate for damage. The fact that the degree of root lesioning was increased in the presence of fungi and nematodes supports this. If the experiment was conducted for longer than 10 weeks significant growth reductions would probably have occurred when either nematode was present with M. bolleyi or F. acuminatum. This work clearly demonstrates that there is an interaction between both nematode species and root rotting fungal species investigated. The fact that South Australian cropping regions contain over 19 different species of fungi (Taheri et aL, Igg4) suggests that further investigations are warranted into the role of the root rotting fungi on the damage caused by either nematode. 185 Chapter l0 Final Discussion and Conclusions Chapter 10 Final Discussion and Conclusions The work described here shows that the root lesion nematode, P. thornei can be considered an economically important pathogen on wheat in South Australia. The damage caused by the nematode in the field is a function of the initial density in the soil and the influence of the environment on the host and the nematode. The geographical distribution of both P. thornei and P. neglectus is widespread in South Australia with a 9OVo chance of finding one or both nematodes in any soil sampled from the cropping regions. P. thornei has a tendency to be found in clay soils, while the closely related species P. neglectus is more commonly found in soils of a sandy composition. However, this distribution is not definitive, as both nematodes were found in all soil types. Plants of commonly cultivated South Australian legumes (grain/pasture), cereals and weed species were sampled from the field, and contained P. thornei and/or P. neglectus. This was expected given that P. thornei is reported to be polyphagous and able to develop on a wide range of botanical families. Aseptic laboratory tests screening a number of cereals and non-leguminous hosts for their ability to support multiplication of P. thornei indicated a range of susceptibilities within genera of the Gramineae. In general terms, wheat is considered to be a 'good' host supporting high P. thornei multiplication. Triticale, rye, barley, oats and durum are 'moderate' hosts with moderately low to moderately high P. thornei multiplication, while the non-leguminous hosts of linseed and canola are 'poor or non-hosts' with negligible nematode multiplication and possible resistance. Similarly, P. neglectus multiplied more on wheat than on either rye or oat cultivars. Although both species of the root lesion nematode were found to have similar reactions to hosts between plant genera, some varieties/cultivars within plant genera (particularly the Gramineae) supported very different rates of multiplication for the two species. The variety 4US4930, which is 186 Chapter l0 Final Discussion and Conclusions resistant to CCN, was one of the least susceptible wheats tested for P. thornei, but the most susceptible for P. neglectus. An assay for screening cereal and non-leguminous hosts to P. thornei was developed and found more rapid, accurate and cost effective over a two month period in a controlled growth room as opposed to five months in an exposed glasshouse. The optimal conditions for this two month assay included growing plants in small polyethylene tubes with sandy soil in a growth room at20"C, and inoculating with a non-damaging initial inoculum density of 400 P. thornei per plant one week after sowing seedlings. Even though experimental conditions for each assay were relatively standardised, care should be taken with the interpretation of results when comparing between assays because plants were inoculated with different inoculum cultures, albeit prepared by the same method. As a consequence, the most reliable interpretation of results was found from ranking the va¡ieties rather than considering actual numbers. Further understanding of the variability in inoculum cultures is necessary. The population dynamics and field relations of P. thornei on wheat were examined both in the laboratory and in the field. Laboratory studies to assess host damage in an enclosed manipulated environment using only the plant and the nematode were inconclusive. Repeated experiments using slightly different experimental techniques to investigate yield relations with P. thornei and wheat differed in outcome, reinforcing the need for a greater understanding of the relationship between the nematode and the host. Smaller container size and sandier soils were associated with higher P. thornei penetration of wheat roots. In almost all experiments, data was obtained by extraction of nematodes from roots over a defined time period. Estimates of the rate of extraction over four days showed that P. neglectus left roots significantly faster than P. thornei, even though similar numbers of nematodes were in the root systems. This further stresses the notion that care should be taken in interpreting data when comparing similar experiments for the two species of Pratylenchus. It is important to have a better understanding of the 187 Chapter l0 Final and Conclusions ecology of. P. thornei. Studies with other Pratylench¿rs spp. have demonstrated the nematode spends time evenly between the root system and in the surrounding soil environment. The question of possible ectoparasitism and the associated damage to root tissue are important considerations, neither of which were addressed here. However the comparable ranking of a range of cereal and non-leguminous hosts was found irrespective of whether nematodes were extracted from roots or soil in combination with roots supports the methodology used in this work. The laboratory studies with wheat and P. thornei suggested that low initial densities, particularly at the early stages of growth up to five weeks after inoculation, result in stimulation of a number of plant growth variables. However, at higher initial densities, particularly over longer time periods, evidence of significant reduction of many growth variables was found. The significant reduction of nodal roots and number of leaves of the main tillers of the wheat plant at high initial densities (15-20,000 P. thornei per plant) would severely affect final grain yield. This affect would be worse if water were limited in the latter parts of the season. The nematode population dynamics found in the laboratory confirmed the basic population studies reported in Ch 2.9. In summary, with low initial population densities, P. thornei multiplication was high, however where the initial density was high multiplication was severely restricted. In all laboratory experiments, the nematode failed to reach "equilibrium density", which was probably due to nonlimiting availability of food and the short duration of experiments. Field population dynamics of P. thornei were assessed over two years in the Barossa Valley at Tanunda and showed that the nematode could severely restrict the yield of wheat in South Australia, depending on the initial density present. Manipulation of field populations was successfully achieved by the use of different cereal and non-leguminous hosts of varying susceptibility to P. thornei. The variation in initial nematode density identified across a given paddock suggest it may be possible to conduct similar studies without such manipulation. The ranking of susceptibility of varieties in relation to their 188 Chapter I0 Final Discussion and Conclusions multiplication, based on soil numbers at the start of the first season and again at the opening of the following season, was not significant but revealed similar trends to the laboratory assay. This supports the value of laboratory-based studies which are conducted in a confined environment avoiding many of the unknown field variables. However, when plants were sampled during the growing season and nematodes were extracted from roots alone, the results inconsistent to those where the soil extracts were taken to establish initial densities at the start of the first season and final densities at the beginning of the second season. This may be due to confounding effects of initial nematode density on subsequent multiplication. Hence, sampling soil with roots at the start of the season was found to be a more accurate way of assessing initial nematode densities. The initial density associated with damage was seasonally variable. In the first year, grain yield of Spear and Machete were reduced by 38 and2T%o at high initial densities of 1506 and 3127 nematodes per 2009 OD soil. However, in the second year of the trial, a reasonably dry year by comparison, the closely associated cultivar rWarigal was found to be reduced by 27Vo at much higher initial P. thornei densities (9000 nematodes per 2009 OD soil). It is possible that the tolerances of the wheat varieties used over the two years were different, but the majority of the variation was probably seasonal over the two years of the trial. Such population shifts over different seasons with initial nematode density responsible for economic damage are reported by other nematologists. As a consequence and in order to use correct information to predict future nematode populations, estimate possible crop damage and suggest management strategies, data on nematode numbers need to be averaged over several years and locations. The levels found at Tanunda over the two year trial are generally higher than those in P. thorn¿i studies from NS'W and Queensland. The two varieties selected by laboratory screening for possible resistance to P. thornei were assessed for their resistance in the field. Both GS50A and AUS4930 had good 189 Chapter Ì0 Final Discu.r.rion and Conclusions field resistance with a multiplication rate of less than one over a season. However, Warigal, a commonly grown South Australian wheat variety was found to have a multiplication rate of 3.88 over the season and also was intolerant. This supports the ranking of host efficiency of wheat cultivars from laboratory screening (Ch. 5), therefore making, warigal, Machete and spear,less favourable wheats to cultivate. Visually, the tillering capacity and plant height of these intolerant, susceptible wheat varieties showed a trend to be reduced at higher initial nematode densities, but this was non-significant. All wheat varieties assessed, except AUS4930, had characteristic root lesioning, which was accentuated at higher initial densities. Lesioning was seen only during the later parts of the season after head formation and was worst at grain filling. High nematode densities were generally associated with a reduction in nodal root development and a reduction in finer laterals on both the seminal and nodal root system on susceptible hosts. This late season effect of P. thornei on the host might be a consequence of the biology of the nematode. V/ork in NSW suggests that low soil temperatures early in the season do not favour nematode multiplication, but as the temperatures rises later in the season (around October) were associated with considerable multiplication. As mentioned the wheat AUS4930, showed field resistance, and superior field tolerance to P. thornei as well as carrying a single gene for Cereal Cyst Nematode resistance. The potential P. thorn¿l resistance of AUS4930 demonstrated by the field trial warranted investigation of the resistance mechanisms and genetics of resistance. Studies revealed that the resistance in both GS50A and AUS4930 occurs post penetration, because similar numbers of P. thorn¿i entered susceptible and presumed resistant hosts. The implications of nematode resistance acting post penetration is of concern for the South Australian cereal growing regions, given the high number of primary and secondary root rotting fungi which are prolific in most cropping regions which may have synergistic interactions with Pratylenchus. The segregation of F2 populations from the cross AUS4930 and the 190 Chapter l0 Final Discussion and Conclusions susceptible wheat Schomburgk suggested that the P. thornei resistance was controlled by either of two independent recessive genes. However, the data should be interpreted with considerable caution due to the overlapping distribution of both parents. This is not surprising given that AUS4930 is a landrace variety, obtained originally from wetter regions of Iraq and was expected to show some variability. It was, surprising that the commonly grown wheat cultivar Schomburgk had some seed which grew into plants with a high degree of resistance. This could suggest that if some re-selection of intolerant, susceptible commonly cultivated wheats occurred, resistance to P. thornei may be obtained directly from highly adapted agronomically favourable current varieties. This was the case with the cultivar GS50A in Queensland, which was re-selected from the highiy susceptible, intolerant wheat variety Gatcher (J. Thompson, pers. comm.). In order to clarify the results, further re-selection ofboth resistant and susceptible varieties is necessary to obtain isogenic lines, followed by a repeated genetic study. Possible association of the nematodes P. thornei and P. neglectus with root rotting fungi was examined in a preliminary study. The fungi Fusarium acuminatum and Microdochium bolleyi were selected for this study due to their similar biology, being late colonisers and cortical invaders similar to Pratylenchus. There was a significant correlation between both nematode species in association with the two fungal species, with a combination of both organisms causing greater damage than when either species of nematode or fungus is present alone. Again, in these experiments, both nematodes were found to be damaging alone, re-confirming the nematodes as pathogenic in their own right. As noted in laboratory studies, looking at wheat yield in relation to increasing nematode density, plant growth was stimulated at low densities, both with and without fungi. At high initial densities, and with time, the nematodes alone and in combination with fungi were found to damage a number of plant variables measured. This initial investigation requires further studies on the association of other fungi with the nematode and their possible role in breakdown of nematode resistant varieties are required. 191 Chapter l0 Final Discussion and Conclusions Commonly, both P. thornei and P. neglectus occur together in the cropping regions of South Australia. They appear morphologically similar to the field nematologist and are difficult for the non-taxonomist to identify using light microscopy. Both nematodes have been described from other countries but no such descriptions are available for Australia. Morphological studies from both f,reld and culture specimens in South Australia show that both nematodes from local populations are comparable to previous descriptions from overseas. The two nematodes are distinguished primarily by the position of the vulva using the light microscope. This study revealed that the percentages used for this distinction may overlap between species, so additional characters should be used to identify species correctly. Use of high power and scanning electron microscopy can allow precise identification of the two species because there are differences in several morphological characters. In addition to the morphological studies, preliminary investigations were undertaken to identify possible molecula¡ differences between the two nematode species. DNA was extracted from both species and digests carried out to identify Restriction Fragment Length Polymorphisms. The digests were probed with enzyme-digested labelled P. thornei. Repetitive band differences were identified between the two species indicating the potential of using DNA markers to allow species delineation. In conclusion further investigations are required on several levels. On the biological level, a more adequate understanding is needed of the behaviour of the polycyclic pathogen and its migration between roots and soil throughout the growing season. The genetics of P. thorn¿i resistance in AUS4930 need to be carefully reassessed, as most plant breeders require single gene resistance for successful breeding programs. Further, the mechanism of the P. thornei resistance is of paramount importance, particularly in south Australia due to the possible fungal associations with the nematode. P' thornei resistance appears evident in the T. aestivum species, but because of the polycyclic nature, polyphagous host range and the population dynamics of pratylenchus, t92 Chapter I0 Final Discussion and Conclusions it is unlikely that rotation with one resistant crop will control the nematode below the threshold for economic damage. The potential resistance of the non-leguminous hosts of linseed and canola needs to be further assessed, but their adaptability and use in South Australia may be limited by environmental constraints. Canola may have great potential as a rotational crop, given that the isothiocyanates it contains may act as a soil biofumigant. Hence, further research to identify other sources of resistance within other cereal or rotational crops used in conjunction with cereals (grain and pasture legumes) is required. Finally, due to the presence of many mixed populations of the two species, and the suggested economic importance of the other species P. neglecttts, an integrated control program will be necessary to manage both species of root lesion nematode. Appendix A 193 Anpendix A: Experimental Data for the Statewide Distribution Survey of P. thornei and P. neglectus in the cereal growing regions. Explanation about data: Counts of nematodes in root samples Column H Root P.thornei I Root P.neglectus J Pratylenchus sp. root total The values have a ranking from 0, low, moderate (mod) and high and refer to the number of nematodes in the root system of the crop sampled (Column G). High > 40,000 nematodes / 3 plants. Moderate 10,000 - 40,000 nematodes / 3 plants. Low < 10,000 nematodes / 3 plants. If a plant has a low, mod. or high rankrng this implies some hosting ability. The ranking however, does not refer to hosting ability but rather simplified the counting procedure in-the laboratory. 'Counts of nematodes in soil samples Column K Soll P.thornei L Soll P.neglectus M Pratylenchus sp. soil total The numbers are all quantitative and refer to the number of nematodes per 200 g oven dry soil. However, because extensive sampling was not performed the numbers should be considerãd u, pr"r"n"" or absence rather than quantitative. Abb¡eviations used in the datå table RBE = Red Brown Earth (red clay soil) - = missing data t APPENDIX A Experimental Data for the Statewide Survey for P. thornei and P. neqlectus in the cereal growing regions Or A B c D E F G H J K L M 1 NO. REGION LOCANON DATE FÂHIIEH SOIL TYPE CUFFEI{T CBOP ROOÎ Þ-lhomC ROOT P.ndlætu. ROOT PET¡9 TOTAI SOIL P-tñomC IOTAL Pht rD SOll. 2 1 5 Lower North Two Wells Oct-92 John ShâÉ low 3 2 5 Lower North Two Wells Oct-92 RBE Wh€dlDúrum) 14 4 3 5 Lower North Two Wells Oct-92 John Shâm Wheá(Durum) 5 4 5 Lower North Two Wells Ocl-92 John Shâ@ orev dry Fù &âns low l0 6 5 5 I ower Norlh Two Wells oct-92 sndv loam Fóa B€âns low 16 7 6 5 Lower Norür Two Wells Oct-92 RBE Orruñ lYallorc¡ì 0.00 80 7 5 I owÊr Norlh I Two Wells Oct-92 RBE Tril¡câl€ 0.00 13 5 Low€r Nofh Two Wells Oct-92 I I FBE Tritical€l&rffi) 0.00 5 I 5 I ower North Mallalla fo Ocl-92 Psl€r Mad bâh Durum fYallåþ¡) low 20 11 10 5 Lorer Norlh Mâllalla Oct-92 P€l€r Mardr RBE low 50 0 50 12 to 5 I owêr Nôrlh Mallalla Ocl-92 sndvlM Pdur€(ba/mdryæÞ$l 12 0 12 13 11 5 Lorer North Mallalle Oct-92 clâv l@m low 60 14 12 5 Lower Norlh Mallalla Oct-92 Pôl6r Mårch loem Mod¡clPar@ilo) low 50 0 50 15 13 5 Lower North Mallalla Ocl-S2 snd Barl€Y lcalhnì low 4 14 5 Lorer No.th 16 Mallalla Oct-92 Psl€r lrish loam Oas(Potoroo) low 10 0 10 17 15 5 Lower Nortr Mallalla Oct-92 sndy loam Pature l8ârld Grassl low 12 0 '12 18 16 5 Lowêr North Mellãllã r)d-92 P€lsr lrish gndv loam PælublHåñâlôrd M.el low 19 16 5 Lorer North Mallalla ocl-92 snû loâm Pætur€{Bãtuv Græsl low 20 17 5 Lower No¡lh Råd Bânks Ocl-92 bhn Blackol RBE 13 21 l8 5 Lowâr North Red Johñ RBÊ Banks ocr-92 Bbckd P€aslAlml low 3 22 19 5 Lower Norlh R€d Banks Ocl-y2 ùhn 8lâcked ¡oam low 40 20 5 I owêr NÕrlh 2t Rod Banks Oct-92 John Blækd Faba B€ãns lFiord) 0.00 40 24 21 5 Lowêr North StockDort O¿t-92 lân Roh& RBE low 10 0 10 25 2it 5 Lôwêr North Stockport Ocl-92 lãn Roh& Paas(Alm) o00 26 æ 5 Lower Norlh Stockoort Oct-92 clay loam 0.00 5l 0 51 27 24 5 Lowêr Norûr StockDorl Ocl-92 lân Rohd€ RBE low 0 0 o 2A 25 5 Lowsr Norûr Tarlee Oct-92 RBF Wh€a(Mæhârsl low I 0 I 26 5 Lorer North Tarlêo 29 Oct-92 Tory Clå*o RBÊ low æ 0 30 27 5 Low€r Norlh TalÊe Oct-92 RBE Chicl@€âlÂmùvll low 12 0 12 28 5 Lower North Tarlæ ô.t-g) 3t Tohv Chtrs RBÊ o.oo 472 0 472 32 2à Lower 5 North Tarlee Oct-92 Tonv Ch*å RBE P€asl^lml low 472 0 472 33 æ 5 Lomr Norlh Tarlæ Oct-92 Graham H¡¡ RBE low 2 34 30 5 Lower Nôrlh Tarlæ Oct-92 GEham Hil RBE Ch¡cþ€å(Amlhvll low 72 0 72 35 30 5 Lorer Norlh Tarlêe Oct-92 Graham Hill RBE Bârål Mådb low 72 0 72 36 30 5 Lowâr Nôrlh qovsr Tarlee Oct-92 caham H¡il RBE Dinn¡nup low 72 o 72 30 Lower Grâhâh H¡ll 37 5 Norül Talæ Ocl-92 RBE low 72 0 72 38 3l 5 Low€¡ Norlh Tarlæ GËham Oct-92 H¡[ RBE Wh€at{Daoqêrì 0.00 8 39 32 5 Lower North Tarlee Oct-92 HBE Pâlursl&l€v/dovåd 3 40 gt 5 Lowêr North Slocknôd Oct-92 D€an Btrson clâv loem Wheal (Mæh€tel 000 0 0 0 34 Lorer 41 5 North StockDort Oct-92 s¡d DurudYâll.¡oil 0_00 0 0 o 42 35 5 Lower North Stockport Ocl-92 O€an Brenson orev clâv Chbkp€å 0.00 0 0 0 1a 36 5 Lower North StockDorl oct-9r2 clav loâm low 6 44 s7 5 Lower Norlh gndv Stockport Oct-92 Brbn Kæh loâm Wìoât(Mæh€l€) low 72 ¿5 38 5 Lower Norlh Stockoorl oct-E2 snd low 2 46 39 5 Lowor Norlh SlockDort oct-g2 Bdan Kæh sndy loam P€âslAlml low 0 0 0 47 ¿l(l 5 Lower North Stockoorl oct-92 Bún Kæh RBE low I 48 41 L¡wer 5 North SlockDort Oct-92 Svd Nã¡n sndvl@ Durur{Yallorci) 000 0 0 0 49 42 5 Lower North SlockDorl Oct-92 Syd Nairn Oals(Maloo) low 50 42 5 Low€r Norlh StockDort oc,.-92 Svd Nåiñ low APPENDIX A Experimental Data for the Statewide Survey for P. thornei andi_lgglgglw in the cereal growing regions rô Oì A B c D E F G H I J K L M 51 43 5 Lower North StockDorl Ocl-92 0.00 0 0 0 52 4 5 Lower North StockDort oct-92 SYd Naim clay loam P€slDirnnl 000 6 o 6 53 ¿15 5 I ôwer North KâDUnde Ocl-92 RBF 0.00 0 0 0 5¿l ¿16 5 Lower North KaDUnda Oct-92 Gery Schmid RBE P€as(Alrol o00 3 55 47 5 Lower North Kâounde o.t-92 RBF 000 14 56 4A 5 Lower No¡th KaDunda Oct-92 AâdsvlClboêil low 57 ¿g 5 LÕwêr North Kaounda oc.-92 RBE 0.00 5 58 50 5 Lower North KaDunda Oct-92 li/h€d {Blåd€) low 59 5l 5 Lower Nofth Kæunde Oct-92 RBE ooo 2 60 52 5 Lower Norlh KaÞunda Ocl-92 MËk Ryan orry clav 000 22 o 22 61 52 5 Low€r North Kaounda Oct-92 Mbk tuañ srìd lÕw 80 0 80 62 53 5 Lower North Keounde o.,-92 RBE Wh6al (Rden) low 2 0 2 63 54 5 Low€r North Kæunde Oct-92 R&d Tllâv RBE low 24 0 24 6¿l g 5 Lower North Kapunda Ocl-92 Robed Tillsy clay loam Wh€d lwâd@l) low '14 0 14 65 55 6 uooer Norlh JamestoM Ocl-92 RBE low 'I 6 66 56 6 UDDer North JamestoM Oct-92 Rob¡n H,a[ Padur€ low 67 57 6 uDDsr North JamestoM Oct-92 LTtumr RAE o00 0 0 0 68 58 6 UDoer North Jamestom Oct-92 M¡cbaloails HBE V€bhlBbrchsllêur) low 3 69 59 6 Uoær Norlh Jâmestom Oct-92 TEvor Fllb FSE 2 70 60 6 llnær Nôrlh od-92 FRF PæluG Túl 0 0 0 71 61 6 Ljooer Norlh Jameslom Oct-92 Trevor ElliE RBE low 2 72 G2 6 lloær Nôrlh James{om Oct-92 RBF Aa&v(Gan€on) low 3 73 6Ít 6 UDær Norlh JamesloM Oct-92 Càris Br€Þa RBE OdslBerdbæll low o 3 3 74 BI 6 uDosr Norlh Jamestom Oct-92 low 75 6rt 6 UDDêr North Jameslom Oct-92 N6v¡ll6 Gb RBE Luo¡nsfcunoaru) 000 0 o o 76 67 6 LJDoer Norlh Jamestom Oct-92 FBE PdublGråsvgovåd 50 77 6A 6 ljoær North Jmês{om Oct-92 GâP Bãth RBE low 26 78 69 6 UDosr Norlh Jameslom Oct-92 Madb Chrke RBE Luo¡nsfDån¡â) low 0 o o 79 70 6 L.,oær Norlh Jameslom Ocl-92 Mãdin Cbrlè c{âv loâm low 0 0 0 80 70 6 UoDer North Jameslom Oct-92 Madh Cbrks $nd low 32 o 32 81 71 6 lJoo€r North Jileslom Oct-92 clev loâm Pdurc lHvkôn Oov6d low 16 62 72 6 llôDêr Nôrth .leñes{ôM Oct-92 Alan Morqan RBE Wh€âtfSÞ€ârl low 2 83 73 6 uooer Norlh Jmestom Oct-92 Bâd OâÞ RBÉ 000 0 0 0 84 74 6 UDoer North .lâmesioM Ocl-92 Malcolm Sparks RBE msalfso€ar) low 51 85 75 6 UDær Norlh JamesloM Ocl-92 Meñn &*å RBE low 5 0 5 86 76 5 Low6r Norür Saddleworth Ocl-92 Mich€alMilhr RBE Bad€vlschoonoil low 24 a7 TI 5 Lowêr North Saddleworth od-92 Mbh€âl M¡lhr RBE low 20 88 7A 5 I ower NÕrlh Sâ.l.llêwôrlh Ocl-92 Mich€al M¡l¡€r R8E BadovlSchoon€r) low 8 89 79 5 Lower Norlh Seddleworfr od-92 MÈh€âlM¡ll6r clãv loem low 44 90 80 5 Low6r Nôrlh Sâddlêwôrth ô.t-92 RßF Vatch(Blarch€ll€url low 4 91 80 5 Lower North Saddleworlh Oct-92 Granlbv McËvov orev clev low 51 92 8l 5 Lower North Seddlêworlh ocr-92 FAE LuÞ¡ns(Guñqaiial 0.00 l0 o to 93 a2 5 Lower Norlh Saddleworth Oct-92 GÉnll€v McEvd s¡l 8!t 5 Lower Norlh Seddleworlh ocr-92 Gtrlhy McEvoy FAE BerclvlGall€on) low 10 95 84 5 Lower North Saddleworlh Oct-92 FAE low 36 0 36 96 &5 5 t ôwêr North Saddleworth ocr-92 Andrew Plwkhehn clav loam Poas(Alm) low o 26 26 97 86 5 Lower Norlh Saddleworlh Oct-92 clev loâm Aâ16þlSkill low I e8 e7 5 I owêr Norlh Sârldlewôrth Oct-92 FAE ChickÞarSafÈnì 10 b 16 99 88 5 Lower North Seddleworth Oct-92 5ræ*htlEl clev loâm 000 6 100 m 5 Lôwer Nôrth Sâddlêwodh õd-92 3r@ Schrual snd Bârl€vlGalloon) 000 o 0 0 APPENOIX A Experimental Data for the Statewide Survey lor P. thornei andÀlg&ctus ¡n the cereal growing regions \o Oì A B c D E F G H J K L M 101 9{t 5 Low€r Norlh Saddleworlh Ocl-92 RBE 0.oo 22 1lJ2 9i 5 Lower North Saddleworlh Oct-92 Greq S€hmâl clev lom Wh€dfKiata) o0o I 103 92 5 Lower Norlh Po¡nt Pass Nov-92 Ron M¡l& clev loam low 0 42 42 1lJ[ 93e 5 Lower Nodh Poinl Pass Nov-92 sndy loam BâdovlGå[ænl low 0 44 44 105 93b 5 Lower North Poinl Pâqs Nov-g2 Ron Mild€ gndv loâm low 0 o o 106 94 5 Lower North Point Pass Nov-92 RBE low 0 24 24 107 95 5 Lower North Pô¡nt Pâss Nov-92 Ron Mìl& sndv loam low 6 108 96 5 Lower North Poinl Pass Nov-92 lryh6at(Dåe€r) low o 6 6 109 97 5 Lower Norlh Po¡nt Pass Nov-92 clav loam low 0 74 74 110 98 5 LowÊr North Point Pass Nov-g2 Whoa(Mæh€t€) low o 1t 1t ttl 99 5 Lower North Po¡nt Pass Nov-g2 John Fåiley low 112 100 5 Lower Norlh Poinl Pas Nov-92 gndy loah Wh6âllMolin6ux) low o 0 0 113 101 5 Lower Nolh Po¡nt Pas Nov-g2 Bob Lsdiþhk€ snd low 0 45 45 114 101 5 Lower Norlh Po¡nt Pass Nov-92 9nd V'/h€d{Sæar) low 0 45 45 115 102 5 Lower North Po¡nt Pass Nov-92 Bó L€dhshks gndv loam low 5 116 103 5 Lower North Po¡nl Pas Nov-92 Bob Ldilshka snd Palur€l8arl€v Græs) low 0 34 34 117 104 5 Lowêr North Po¡nt Pass Nov-92 Ross Schü snd low 2 118 105 5 Lower North Point Pass Nov-g2 sody loam !{¡1ì€aÎ(Mol¡n€ux) low o 300 300 11S 105 5 Lower Norlh Point Pass Nov-92 Ro$ Sch& gndvlM low 0 300 300 124 105 5 Lower North Po¡nt Pass Nov-92 low o 300 300 121 106 5 Lower North Poinl Pass Nov-92 Ros Schû $ndv loam low 0 0 0 122 107 5 Lower North Po¡nt Pass Nov-92 snd low 0 5 5 123 108 5 Lower North Po¡nt Pass Nov-g2 snd WhsallSoeâr) low o 112 112 124 109 5 Lower Norlh Po¡nt Pas Nov-92 Go6í Shce gndv loâm 3 125 110 5 Lowsr Norlh Point Pass Nov-g2 Gáóf $hú7 Palur€lBarl€y qrasdclwsr) 3 12 ttl 5 I owêr Nôrlh Po¡nl Pãss Nov-92 Mabom kh@ end 0 66 66 127 112 5 Lower North Po¡nt Pass Nov-92 Maholm $hú clev loam low 0 4 4 12A 113 5 I ôwer Nôrlh Po¡nt Pãss Nôv-92 Mebolm Sc@ e¡dv loam P€as(thnn) lôw 0 12 12 12S 114 5 Low€r North Po¡nt Pass Nov-92 Mãholm Sh@ snd low 0 6 6 130 115 3 ÊeslÊm Evre Kimbã Nôv-93 Richard PaBons RBE Pålur€ fmdb) 0 low low 0 486 ¿l86 131 116 3 Eastem Evr€ Kimba Nov-93 RBE 0 low low o 74 74 132 117 3 Eestern Evrê Kimbã Nov-93 F¡clÉrd PesroÉ RBE Wheat lMschelel 0 5300 5300 133 118 3 Eastern Evr€ Kimba Nov-93 PolåÌ &¡nkå 0 3815 3815 134 lt9 3 Eãslârn Evrê K¡mbâ Nov-g3 Pet€r B€¡nk€ qrev clav 0 low low 0 650 650 135 119 3 Eastern Evrê K¡mba Nov-93 Pâlâr &inkâ 0 low low o 650 650 136 119 3 Eestem Evre K¡mbâ Nov-g3 Pet€r bê¡nk€ qrev clav PâslurelHâó¡nosr Medicl 0 low low 0 650 650 137 120 3 Eastem Evre Kimba Nov-g3 Pêlêr &¡nk€ læm Wheat lSFar) o 650 650 138 121 3 Eestem Evre Kimbâ Nôv-93 All€n ssmpson RBE Pastur€lCal¡oh Med¡c) 0 low low 0 't000 1000 13S 121 3 Easlem Evre K¡mba Nov-g3 Allsn sammon RBE Pâslù16lHârbiñdêr M.d¡cl 0 low low o 1000 1000 140 122 3 Eastem Evrs K¡mbe Nov-93 clay loam Bâil€v {Gâll€on) 0 662 662 141 123 3 Eastem Evre K¡mbâ Nov-g3 Fod Ljsneil RBE 0 mod mod 0 3130 3130 142 124 3 Eestern Evre Kimba Nov-93 RBE Wh€at (Mâch€l€) 0 992 æ2 I ó3 126 3 Easlern Evre Kimba Nov-g3 Dâån Willbms RBE 0 12AO 144 126 3 Eestem Evre Kimba Nov-g3 FBE Wh€åt lSæar) 0 97 97 145 127 3 Feslern Fvrc Cleve Nov-g3 R@er Neild clâv loem o 3A 38 146 124 3 Easlem Evre Cleve Nov-93 R@êr Nê¡ld sñdv loil Oals (Marlool 0 70 70 147 129 3 Easlem Evrs Cleve Nov-93 Ræer Ns¡ld RBE Clov€r (Paradana Bâlencia) o 66 66 148 130 3 Easlem Evre Clêvê Nov-93 F@€r Neild RBE Pâslure 0 5 5 l4s 131 1 Westem Evre Smokv Bav Aor-93 Bill Hußon snrl Oals (Wallaræ) 0 292 æ2 150 132 I Weslern EvÉ Smokv Bâv 40tr93 Elll Blureon snd 0 140 140 APPENDIX A Experimental Data for the Statewide Survey for P. thornei and P. neglectus in the cereal growing regions l'-' o\ A B c D E F G H I J K L M 151 133 I Westêm Evrê Smokv Bãv Anr-93 Bill Blußon 9nd Påslurc 0 312 312 152 134 1 Westem Evre Smokv bav 4Dtr93 tl¡ll Blurcon snd &lev fGáll6onl 0 u7 u7 153 135 I Wêsleh Ev.â Smokv flev Aor-93 snd Påsfurê 0 27 27 1s4 136 1 Westem Evre Smokv Bav 4Dtr93 B¡l Slureon sÐd o 17 17 155 137 1 Westeh Evr€ Smokv Bâv Aor-93 Bi[ glußon snd Pâslur6 0 86 86 156 138 1 Western Evr€ Smokv Bav ADr-93 Ell Blumson snd 0 836 836 t57 t 3!¡ 'I Westem Evre Smokv Bav Jul-93 sand Peslure 0 890 890 158 f40 1 Western Evre Smokv Bav Jul-93 Bi[ Blumon snd 0 1465 1465 159 141 1 Western Eyre Smokv bâv Jul-93 Bill Blußon snd Psslure 0 1 140 1 1¿10 160 142 I Western Evre Smokv Bev Jul-93 Biil Blumson snd 0 2AO 280 t6t 143 1 Weslern Evre Smokv Bav Jul-93 El¡ Blußon snd PaslutÊ 0 2660 2660 162 144 I South East Bordertown Jul-93 Slâôhen Hole sfftu lôâm 0 198 198 163 145 9 South East Borderlown Jul-93 St@hen Holg snd Paslür€ 0 0 0 164 146 I South East Bordertown Jul-93 Slmhân Holè 0 65 65 165 147 I Murrav Mallæ Cambra¡ Jul-93 Norm W€¡nk€ snd &rlêv 0 25 25 166 148 3 Easlêrn Evr€ K¡mba Oct-93 RBE Medic fPårab¡nqa) 0 low low 0 1215 1215 167 l¿9 3 Eâslern Evrê K¡mbâ Oct-g3 D€an W¡lliams RBE Wh€al lsæåd 0 low low 0 2690 2690 168 150 3 Eastern Evrs Kimba Oct-93 D€an Will¡ams FBE o low low 0 I 104 1t(x t 6s t 5l 3 Eestêm Evre Kimbâ O.J-93 DeEn W¡ll¡ems BBE Wheat {Mach€t€) 0 low low o 3500 3500 170 152 1 Westem Evre Smokv Bav Oct-93 Bill Blureon snd 0 'ls2 192 171 153 I Westem Evfg Smokv bav Oct-93 Bill Blußon 0 1800 1800 172 l5¿l I Wês{êm Evrê Slrêekv bâv Oct-93 Don G.6¡q snd Wheel {Jenzl 0 low low 0 q 40 173 155 'l Weslêm Evre M¡nniDa Oct-93 Mhnba RC clâv loâm o low low 0 4000 ¿lOOO 174 56124 1 Westem Evro M¡ñn¡Dã O.J-g3 Bruc€ H€ddle clåv læm Cånoh 0 hioh h¡oh 0 5000 5000 175 ts7 1 Weslern Evre St€akv Bav Oct-93 Don Gßo clev loâm 06ls o h¡oh hiqh 250 250 5{X) 176 158 I Wêslêm Evrê M¡nn¡m ô.t-93 Mhnia FC clav loam Chicb€a 0 mod mod 0 u u 177 59124 1 Weslern Evre M¡nn¡Da Oct-93 B.uc€ H6ddlê clav loem 0 hiqh hiqh 0 986 986 178 6l¡l2tl I WêsîÞm EvE M¡nn¡oâ ô.J-93 clav loam canola (Nsr€ndral 0 h¡oh hioh o 3135 3l 35 17S 161 3 Eastem Evre Cleve Nov-g3 MV Båmmenn clav loem 0 6t 6l 180 162 3 Eestem Evre Clevê Nov-g3 clav loam Paslur€ o 68 68 181 163 3 Eastem Evre Cleve Nw-g3 dev loem Bârlêv lGålleonl 0 156 156 142 164 3 Eastem Evre Clev€ Nov-93 chy loam Paslurg 0 ¿165 465 183 165 3 East€m Evre Cl€vs Nov-93 T¡m Krâehs clåv loâm Wh6ål lMåchel6láñ7ì 0 86 86 184 166 3 Eâslem Évr6 Clêva Nôv-q3 Tim Kra€h€ clav l@m Wheåt (Mechet€l 0 32 32 185 167 3 Eastem Evre Cleve Nov-93 Tim Krâ€h€ clev loem o low low 0 95 95 186 167 3 Eastem Evre Cleve Nov-g3 clav lom Brore Gress 0 low low 0 95 95 1e7 l6a 3 Fedem Fvrê Clev€ Nov-g3 D€nnis F¡€qâd clav loam 0 212 212 188 169 3 Eastem Evr€ Cleve Nov-93 John Ranlord clâv loâm 0 460 ¿160 18S 170 3 Eestem Evre Clêv6 Nov-g3 John Banford clav loil Wheal lJanz) 0 4'15 415 190 171 3 Eastsm Evre Clev6 Nov-g3 John Ranlord sndv lôam asturê lPârâb¡ñdâ/Hãó¡ñd. 0 375 375 191 172 3 EasÌern Evfe CÆrell Nov-g3 sndyl@ Wheal(Machete) 0 1050 '1050 192 173 3 Easlern Evre Cowell Nd-93 W¡ll¡åms sndvlffi m€al(Excal¡bur) 0 low low 0 11 11 (ìôwêll 193 174 3 Eestem Ev.6 Nôv-g3 Gæf P¡oqot HBE Pasture 0 374 374 194 175 3 Easlem Evre Cowell Nov-g3 Gêôf P¡doõl Paslur€ 0 45 45 195 176 3 Fâslêrn Fvre Cowell Nw-93 Geof P¡oool sndv loâm 0 I I 196 177 3 Eestern Evre C¡well Nov-g3 sndv loam Wheal(Jenz) 0 20 20 197 17A 3 Eastern Evre Cowell Nov-93 Rooff SloN sndv loem 0 250 2s0 19ß 179 3 Easlem Evre Cowell Nov-93 sndv loam Barlev(Schooner) 0 u 64 19S 180 3 Eastern Evre Cowell Nov-93 Rooer Noris clâv bem Wh€at{Schomburqkl 0 20 20 2fJ4 t 8t 3 Eeslêm Evre Cowâll NÕv-93 B@er Norr¡s clev loam Wh€atlSchomburokl 0 low low 0 25 25 APPENDIX A Exper¡mental Data for the Statewide Survey lor P. thorne¡ and P. neqlectus ¡n the cereal growing regions æ o\ A B c D E F G H J K L M 201 192 3 Eastern Evr€ Cowell Nov-93 Fæer Noris clav loam 0 38 38 202 183 4 Lower Evrs KâD¡nnie Nov-93 AD t ML Ne$ sndy þåm 8ål€v(schooner) 0 0 0 2fJ3 1A4 ¿ Lower Evre Kæ¡nnie Nov-93 AD & ML Nâss sndv loâm 0 14 14 204 185 4 Lowef Eyre Kâoiñn¡ê Nov-g3 AD E ML Nes srÉv loan WheâtlSchomburql) 0 low low 0 o 0 205 186 4 Lower Evre KaDinn¡ê Nov-g3 AD å ML Ness sndv bem CanÕlãlEu¡dkel o low low 0 0 0 206 187 4 Lower Eyre Cuñmins Nov-93 sndy l@m Wh€ållYarålinka) 0 low low 0 49 49 2fJ7 188 4 Lower Evre Cumm¡ns Nov-93 D€an FG clãv loam Bal€vlChsb€c) 0 36 36 208 189 4 Lower Evre Cûmm¡ns Nôv-q3 clav loam Wh6atlSÞ6ar) 0 low low o 1550 1550 20s 190 4 Lower Evre Cummins Nov-g3 Gordm Modrâ clev loam WhêâtlS¿homburdk) 0 low low 0 80 80 210 191 4 Lower Evre llnõ^ÍJe Nôv-93 F*e Gale clâv loam Wh€al(Janz) 0 mod mod 0 498 ¿98 211 192 4 Lower Evr€ Unoarfa Nov-g3 FæGab dâv loem 0 42 42 212 193 4 Lower Eyre lJnoãrre Nôv-93 day loâm Whest(Janz) 0 130 130 213 194 4 Lower Evr€ Unoarfa Nov-93 Ræ Gal€ dev loam WhealfJânz) o 167 167 214 195 ¿ I ôwer Evre GrænDelch Nôv-93 0 low low 0 2A 2A 215 195 4 Lower Evre GfeenDatch Nov-93 Ph¡l Hvd€ sndv loam PãsturelHalum tuådrãssl o low low 0 28 28 216 196 4 Lôwêr Evrê Ga€enDâtch Nd-93 Pâsture{caÞewe€d) 0 low low 0 81 81 217 196 4 Lowef Evr€ GreenDatch Nov-93 Ph¡l Hvde sr¡dv loam PåsturêlBarlev Gress) 0 0 0 o 81 81 214 197 4 Lowêr Evrê GrænDatch Nov-93 o low low 0 12 12 219 197 4 Lower Evfe Grænpatch Nov-93 Ph¡l Hvd€ endy lom 0 low low 0 12 12 220 198 4 Lower Evre Wen¡lle Nov-93 0 32 32 221 199 4 Lower Evrs Wan¡lla Nov-93 sndv loan Barlev{Schoon€r) 0 o 0 222 200 4 Lower Evrê Cumm¡ns Nov-g3 H¡hon Tr¡oo RBE &rålvlSchoonerì 0 75 75 221 2l)1 4 Lower Evre Cumm¡ns Nov-93 Hillôñ T¡hd 3491 't164 4655 224 202 4 Lowâr Evre GreenDatch Nov-g3 Jack Borlas clav loan P6slur€ o 108 108 225, 203 ¿ I ôwêr Evre GÞêñoetch Nd-g3 o low low 50 50 100 226 204 4 Lower Evre KoDoio Nov-93 Bronlon Grcwd€n sndv l@m Wh€al{Aroona) o o 0 227 20s 4 Lower Evre KoDD¡o Nov-93 0 0 0 228 206 4 Lower Evrs R. L¡ncoln Nov-93 Ashlev Fl¡nl sndY loam Fellow o 26 26 229 2lJ7 4 Lower Evre Tumbv Bev Nov-93 Wh€el(Jânz) 0 114 114 230 208 4 Lower Evr€ Tumbv Bav Nov-93 Pel6a Swefê¡ clav loah o 21 21 291 209 4 Lowsr Evre Yeelana Nov-g3 RBE 40 40 80 232 210 4 Low€r Evr€ Yâêlânâ Nov-g3 P€td Glov€r RBE BarlevlSchoonêr) 2ú 200 400 23? 211 4 Lower Evre Ysâlana Nov-g3 RBE 0 419 419 234 212 4 Lower Eyre Yeelana Nov-93 Davìd Smilh HBE P€eslDundaleì 0 low low 0 tl5 115 235 213 4 Lower Evre Yeelana Nov-g3 David Smilh RBE low low low 115 41 1s6 216 214 4 Lowêr Evre Cummins Nov-93 Berlev 0 80 80 237 215 4 Lower Evre KoDD¡o Nov-93 L€s Schn€¡d€r clav loam WheatfH99Eì 0 low low o 90 90 238 216 2 Central Evß Kvancutte Nov-93 P6t6r OBr6¡n 0 74 74 239 217 2 Central Evfe Kvancutta Nov-93 Petor O'8re¡n sndv bam Påslurg lonion Ws€d) 0 low low 0 54 54 240 21A 2 CentEl Evre Wud¡nnã Nov-93 0 I 105 I 105 241 219 2 Central Evrs Wud¡nna Nov-93 Ken Scholz sndv loam Luoinslcunoerru) + Oeb 0 44 44 242 22f) 2 CentÉl Evr€ Werramboo Nov-93 sndy loam Pasluae 0 270 27lj 243 221 2 Central EvE Warramboo Nov-93 Bråd Schk* sndv loem 0 155 155 244 221tb 2 Centrâl EvE I ôck Nov-g3 Anùew Polk¡nqhqne €lcåræus snd 0 7A 76 245 222 2 Central Evre Lod( Nov-g3 elcármus srìd 0 405 4û5 24G 221 2 Central Evrs Læk Nov-93 Trwor Pierce clav loan Barl€vlCh€b€c) 0 185 185 247 224 2 Cenlral Evre Lod( Nov-g3 clâv l¡râñ 0 244 244 248 224 2 Central Evrs Too qeH Nov-93 Dav¡d Habær sndv lam Yvh€âlfEr@libuil o 293 293 249 226 2 Central Evre Tool¡o¡ê H¡ll Nov-93 Pesture(Haôinq€r) 0 524 524 2sà 227 1 Western Evr€ Wud¡nna Nov-93 R@êr SchoÞ sndv loâm OalslWdlårooì o mod mod o 476 476 APPENDIX A Experimental Data for the Statewide Survey for P. thorne¡ and P. neolectus ¡n the cereal growing regions o\ o\ A B c o E F G H J K L '22A M 251 1 West€m Eyrs Wudinna Nov-93 Ræsr SchoÞ sndv loem Påsture(Fyæ.assl 0 mod mod 0 417 417 252 229 1 Westem Evfe Wudinna Nov-g3 sndy loam Oals(Wallsroo) 0 660 660 25X 1 Westem Eyre Wud¡nna Nov-93 Ræer SchoÞ sndv loem &råtulGellâônì 0 low low 0 265 265 'I 254 231 Western Evre Wudinna Nov-93 sndy loam WheållMach6le) 0 720 720 25â I Wedêm Fvre l ril.l¡nnã Nov-g3 Ræ€r SchoÞ sndv loâm 0 hioh h¡oh 0 4080 4080 256 293 1 Weslern Evrê Wud¡nna Nov-93 Wheel{So€ar) 0 hioh h¡ah 0 1515 1 s15 'l 257 234 Westem EvrÊ Wudinnâ Nôv-93 Roqer Scholz sndy loam M€diclHsrbino€il o low low 0 1170 1170 258 235 I West€m Evre Kvancutta Nov-93 KÊün $hm Rve{SA) 0 low low 0 58 58 259 236 I West€m Evrê Kvencúllâ Nov-93 Kevin Sc@ sndv loam Oab rWelbroo/Ponorool 0 low low 0 516 516 260 237 1 Western Evrê Kvancutla Nov-g3 Barlêv(Galleoñ) 0 low low 0 780 780 261 23A 1 Weslêrn Evß Kvencúllâ Nov-93 K6vin SchopÞ sndv loam BailåvfCh#c) 0 h¡qh h¡oh 0 1812 1812 262 239 1 Weslern Evre Kvancutta Nov-g3 Kâv¡n Schm Wh€al(Machel€) 0 low low 0 565 565 263 240 I Wêstem Evrê Strâkv Bâv Nov-g3 Don G.eh €lcaræus snd Bårl€vlGallmñl 212 6¿8 860 'I 264 241 Wsstem Evrâ Streakv Bav Nov-93 tuñ Gr€b €l€ræus snd Paslurs 0 ¿160 460 265 242 I Wêslâm Fvrâ Strââkv Bâv Nov-93 Don Greh €lcåræus $ard 855 2566 3/.21 'I 266 243 W€stem Evf€ Streekv Bav Nov-g3 tun Greb €lca@us $nd Wh€elfMo¡¡nsux) 30 270 300 267 244 I Wes{êm Evre Slreâkv Bâv Nov-g3 Don Greiq €lcaercus snd 333 1665 r998 268 245 1 Weslern Ev€ Streakv Bev Nov-93 Doh GrB¡d Paslurs 184 551 735 269 246 I Weslêrn Evrâ Slreekv Bâv Nôv-q3 Don G.€¡a øl€ræus snd OålsfPolorÕo) 0 47 47 270 2|7 1 Weslsm Evre Streakv Bav Nov-93 Don Grêb ahårsus srd 100 40 14n 'I 271 244 Westem Evre Streakv Bav Nov-93 Brucê H6ddl€ clav loil WheallSchomburdkì 0 252 252 272 2¿9 I Wêslêm Evrê M¡nn¡pa Nov-93 8ruc6 H€ddl€ clâv loem 0 low low 1500 1500 3000 273 251 1 Weslern Evre Minn¡oa Nov-g3 clav loam Paslur€{Bådêv Grassì 250 926 1176 274 252 I Weslâm Fvre M¡nn¡pa Nov-93 Bruc€ Heddle clav loåm WheâllSchôhbùrdkl low low low 4500 4500 9000 275 253 1 Welsm Evre M¡niDoa Nov-g3 clay loam Wh€alfBf Schomburok) 0 low low 0 425 425 276 254 I Weslem Evß Minniôâ Nov-93 &uæ Hddls sndv lom Peslur€lRv6râssl mod mod mod 0 456 456 277 1 Westem Evrê Minn¡Da Nov-93 &uæ Hddk esturs(Parab¡nqer/Harbinoe low low low 357 1071 Á24 278 256 7 Yorke Me¡tlend Nôv-94 R@6r Johns sndv loam Pâstur€fHsrb¡noâd mod mod mod 6 50 56 279 257 7 Yorkê Maitland Nov-94 RæÊr Johns clãv lôãm Med¡c{Moqul) low low low 26 27 53 280 2s8 7 Yorkê Maitland Nov-94 HBE MediclKâl¡oHBuril lt 11 22 2A1 259 7 Yorks Maitlild Nov-94 Brondon Malon€v RBE Ch¡ckæås(Kåhiva) 0 hioh h¡oh 18 l8 36 2A2 259 7 Yorfts Ma¡tlend Nov-gil RBE L€nt¡1155881 0 low low 18 l8 36 7 Yôrkê 2A3 259 Maitland Nov-94 Brendon MÊlonev clâv lom 0 low low 12 12 24 284 260 7 Yorke Arthurton Nov-94 clav loah Linole lEvr€) 0 0 0 0 0 0 285 261 7 Yorke Ma¡lland Nov-94 Clinlon K¡ilo clav loâm 0 0 42 266 262 7 Yorke Maitlend Nov-94 T¡âvor Polk¡ndhôhê clay loam Cânols(Dunkeldl mod mod mod 120 69 189 247 262 7 Yotke Mâillând Nov-94 Tr€vor Polkinqhorn€ clav loâm Muslârd lEbonvì low low low 'l05 52 157 2Aâ 263 7 Yorko ïoara Nov-94 RBF Peas lAlma) 0 l0 10 289 264 7 Yorkê Yorke Town Nov-94 L6ith Dsn¡sl s¡ltv sìd low low low 290 264 7 Yorke Yorkê Town Nov-94 Lêifr Den¡êl snd M6dicf56r€na) low low low 291 265 7 York€ Waræka Nov-94 J Koennecke snd Medic(Paraænlo) 0 mod mod 6 't8 24 292 266 7 York€ War@ka Nov-94 snd B€d€vlFrånklinl 0 mod mod 0 4i) 43 293 2Ê7 7 Yotke M¡nlaton Nov-94 F¡óârd Jtrre¡n dâv krem Ch¡ckÞeaslD€siv¡cl 0 low low 12 4A 60 294 267 7 Yorke M¡nlaton Nov-94 Ridìard J€rm€¡n clay loam Lalhurusln€w lmumâ) 0 0 0 12 50 62 295 268 7 YÕrkê Curamulka Nov-94 Gordon Stons Pasb¡€ 0 70 70 296 269 7 York€ Smdilmds Nov-94 R€x Kåk6chke clây loâm Msd¡clSanliaooì o hiqh hioh 16 72 88 297 7 Yorkê 269 Sandilands Nov-94 F€¡ Kak6chke clav lom Med¡c(S€ph¡) 0 hioh h¡dh 16 72 88 298 269 7 Yorkå Smd¡lands Nov-9¡l clav loil Med¡clSaæì o h¡qh hioh 16 72 88 299 269 7 Yorkê Sand¡lands Nov-94 B€x Kak6chk€ clav loem M€d¡c(Perabinqa) 0 h¡oh hiqh 12 51 63 300 270 7 Yofte Ma¡tland Nov-94 clâv loam Coriandar o v low v.low 40 16 56 APPENDIX A Exper¡mental Dala for the Statewide Survey for P. thornei and P. neolectus in the cereal grow¡ng reg¡ons o ôl A B c D E F G H J K L M 301 271 7 Yorke Sând¡lân.ls Nov-94 Peul SchuÞo sndv loam Fåba B€ans flærGl o mod mod 31 42 73 302 272 7 York€ Sand¡lilds Nov-g4 Påui shulzå sndv loâm 0 low low 14 70 84 303 271 7 Yorke Ma¡tland Nov-94 day loam Msdic fSanl¡aooì 0 low low 30 50 80 304 274 7 York€ Cunlille Nov-g4 Rod Devi€s sndv loam Mdic lKâl¡oh| 0 low low 124 u 212 305 275 7 Yorkê Kainta¡n Comer Nov-g4 clav loan Pees /ChickD€as 72 192 264 306 276 7 Yorke Kaintain Comer Nov-g4 Ne¡l Pont¡fåx chv loam Cânolil¡nolå 0 low low 20 10 30 30? 277 7 Yorke Ka¡nta¡n Comer Nov-g4 Nêil Ponlif6s endy loam CaonlslFåinbow) 0 low low 30 10 40 308 27A 9 South East Padthawav Nov-94 Wellv Elsden clâv ¡oam ùdând€r 0 o 0 30s 27l¡ I South Eest AoslevlVictoriaì Nov-94 ChÌis Honnff endy loil OatsfCorbEr) 0 0 0 310 280 9 South East ADslsvlVictoria) Nov-94 Chds HonM clâv lom 0 0 0 311 281 9 South East Wolslev Nov-94 Eric Pi€tch clav loam WheållJånzl 350 0 350 312 282 I South East Wolslev Nov-94 Ênc Pielch cliev loâm Wh€åt(J6nz) 350 0 350 313 243 I Sôrúh Fâst Mlrndüllâ Nov-94 Dav¡d Low€ clav loam LuoinslGunoaruì low low low 128 0 124 314 2A4 I South Eest Mundullâ Nov-g4 Whesl(Jenz) 0 100 100 315 I Sôrdh Fãsl Múnafirllâ Nov-g4 Dsvid Low€ clâv loâm 0 170 170 316 286 9 South East Wolsslev Nov-g4 Brucâ &ll¡noer WheellJ6¡z) 150 150 300 317 247 I Sôúth Eâst Wolselev Nov-g4 J¡m M¿dle qrev clãv B€rlêv loh€b€cì 120 n 160 318 248 I South Eæt Kan¡valvictoriaI Nov-94 Â&EDv€Ì low low low o o 0 319 289 I South Eest Borderlom Nov-g4 sndy loam Båley fSchoon€r) 24 0 24 g a2î 290 Sôrih Fâsl Bordertown Nov-94 Mich8€l Llvod€ sndv loam WhêellTãliârâl 0 o o 921 291 9 Soulh East Ken¡va lvlctoriaì Nov-g4 Durum (YEllaro¡) 50 ^¡Etuer 10 60 s22 292 I Sorrth Eâst Kân¡vâ lV¡clôriâì Nov-g¿ A&EDyêr clây loâm Wh€allJånz) 0 0 0 323 293 I Murrav Mallæ Cænmandook Nov-g4 endv k)âm 0 mod mod 0 600 600 s24 294 I Murrav Mallæ Coonmandook Nov-94 sndy lom Wh€el(JânzÌ 0 low low 0 160 160 325 295 I Murrav Malleo Yuroo Nov-94 A Kr€¡o sndv loem low low low 0 o 0 326 296 I Mumv Mallæ Yuroo Nov-94 sndy loãm Belev(Schoon€rl 0 12 12 427 297 8 MurEv Mallæ Coonmandook Nov-94 Marcus Kl€iniq snd Luo¡ns 0 o o 328 294 I MurEv Mallæ Cænmandook Nov-g4 Csnolå(Râinbow) o 450 450 329 299 I MurEv Mallee Yumali Nov-g4 N-þ€l Dåv snd low h¡qh hioh 100 350 450 330 300 I MurÉv Mallæ KiKi Nôv-94 snd garlovlYaosn) low low low 0 0 0 331 301 9 South Easl Bordertom Nov-94 RtMHunt sndy loam Chinâs Gbhoê low low low 0 o 0 332 302 9 South Eest BordertoM Nov-94 R&MHunl clay loam Suqa¡ Peås nof4 low low low 50 0 50 333 303 I SÕrfh Fâst Boialedôwn Nôv-g¿ R&MHunt sndv loam &mmsr&l Onions o o 0 0 0 o 334 304 9 Soúh East Mundulla Nov-93 Chds Lsch ehdv loam BeÊns (lcerus) low low low o 15 15 335 305 I Solnh Eâsl Mrñdullâ NÕv-g3 Chris Læch sndv loam Coriend€r o low low 5 20 25 336 306 I South East Mundulla Nov-g3 Chds Læch Wheel(Ms€r¡nq) 0 low low o 0 0 337 307 I Soúlh Eest Bùck¡nohem Nôv-93 N€vill€ Ws¡ss endy loam Canola lDunk€ldì 0 0 o Appendix B 201 Appendix B: Experimental Data for the Fietd Population Dynamics and Yield Relations of P. thornei on cereals for the2 year trial at Tanunda. Explanation about columns: Column Abbreviation Explanation A Plot No. Plot Number (from 1...130) B v93 Variety grown in 1993 C Rep93 Replicate number (there were 10 reps for each variery) D YTH93 Yietd of individual plot in 1993 (tonnes per hecrare) E v94 Variety grown in 1994 F YTH94 Yield of individual plot in 1994 (tonnes per hecrare) G ID93 Initial P. thornei density 1993 @er 2009 oD soil) H ID94 Initial P. thornei density 19941per 2009 oD soil) I ID95 Initial P. thornei density 1995 lper 2009 oD soil) J MR93 P. thornei Multiplication rate over the 1993 season K MR94 P. thornei Multiplication rate over the 1994 season * Missing data Appendix B 202 PLOTNO. V93 IREP93 YTH93 v94 YTH94 lD93 tD94 lD95 MR93 MR94 1 Machete 1 3.61 GS50A 1.6s 124.18 995.84 1276.58 8.O2 1.28 2 Spear 1 4.24 AUS4930 | 1.35 1285.71 5920.88 295s.63 4.61 0.5 3Warioal r14.42 Wariqal 1.91 101 .1 419.45 3418.61 4.15 8.15 4 Grimmett: 1 3.27 AUS4930 1.47 40.66 202.78 609.89 4.99 3.01 5 Currency 1 Wariqal 1.76 57 .14 55.56 30.87 0.97 0.56 6 Linseed 1 GS50A 1.18 87.91 84.72 598.78 0.96 7.O7 I 7 Fallow 1 Wariqal 1.38 42.86 52.78 65.43 1.23 1 .24 8 Molineux 1 ,4.66 GS5oA 1.46 87.91 429.17 1996.35 4.88 4.65 9 Canola i 1 0.57 Warigal 1.66 18.68 54.17 207.41 2.9 3.83 10 ',t Yallaroi 1 3.42 GS50A 1.5s 85.71 52.71 63 5.82 1 .7I 4. 1 6 11 Echidna 1 4 GS50A 1.42 35.16 543.06 493.84 15.44 0.91 12 Tahara 1 3.83 GS50A 1.61 108.79 166.67 346.92 1 .53 2.08 1 3 GS50A 1 3.1 1 AUS4930 1 .1 3 36.26 256.95 1039.53 7.09 4.05 14 Fallow 2 Warioal 1.45 20.88 21 't .11 503.72 10.1.1 2.39 15 Tahara 2 4.58 Wariqal 2.05 23.08 81 .95 265.44 3.55 3.24 16 Warioal 2 4.52 Warisal 2.16 21.98 81.95 492.61 3.73 6.01 17 Yallaroi 2 4.3 Wariqal 65.93 23.61 67.9 0.36 2.88 18 Linseed 2 0.79 Wariqal 1.81 26.37 31 .94 53.09 1.21 1.66 19 Currency 2 3.62 Warigal 2.06 38.46 40.28 181.49 1.05 4.51 20 Machete 2 3.42 Warigal 2.3 882.42 1088.9 3022.3 1.23 2.78 21 Grimmett 2 3.57 Wariqal 534.07 1369.46 733.35 2.56 0.54 22 Molineux 2 3.88 GS5OA 405.49 438.89 1358.06 1 .08 3.09 23 Spear 2 4.8 GS50A 1.42 478.02 1177.79 1088.92 2.46 0.92 24 GS50A 2 2.92 Wariqal 1 .7 4 306.59 313.89 2111.17 1.02 6.73 25 Canola 2 0.34 Warioal 1.44 150.55 500 938.3 3.32 1 .88 26 Echidna 2 3.97 GS50A 1.39 25.27 288.89 1902.52 11.43 6.59 27 Yallaroi 3 4.51 Wariqal 1.62 29 .67 12 .5 98.77 0 .42 7 .9 28 Currency 3 3.23 GS50A 1.35 21.98 81.95 283.96 3.73 3.47 29 Grimmett 3 AUS4930 1 .3 60.44 15.28 907.43 0.25 59.4 30 Canola 3 0.4 Warioal 1.7'l 7 5.82 1 73.61 469.1 5 2.29 2.7 31 Machete 3 3.84 Warigal 1.77 47.25 311.11 't481 .52 6.58 4.76 32 Molineux 3 3.66 Warigal 1.85 51 .65 480.56 1083.98 9.3 2.26 33 Tahara 3 3.63 Wariqal 1.74 7.69 79.17 1228.43 10.29 15.52 34 Spear 3 4.27 Warioal 1.32 284.62 5200.04 17719 18.27 3.41 35 Canola 3 0.46 GS50A 1 .14 128.57 2555.62 36 Echidna 3 4.02 Wariqal 1.87 81.32 300 203.71 3.69 0.68 37 Linseed 3 0.77 Warigal 1.85 16s.93 184.72 232.1 1.11 1.26 38 Warisal 3 4.24 GS50A 1.65 131.87 369.45 834.59 2.8 2.26 39 GS50A 3 3.02 Warigal 2.05 51.6s 270.84 966.69 5.24 3.57 't 40 Spear 4 4.22 Wariqal 98.9 1 8.06 1 133.36 1 .1 9 9.6 4'l Grimmett 4 3.28 GSSOA 58.24 143.06 1334.6 2.46 9.33 42 Linseed 4 O.52 Wariqal 1.68 112.09 140.28 1712.39 1.25 12.21 43 Echidna 4 3.85 Warigal 2.07 282.42 843.06 888.91 2.99 1.05 44 GS50A 4 2.72 AUS4930 1.29 71 .43 195.83 750.64 2.74 3.83 45 Warisal 4 3.72 AU54930 1.38 123.08 1 105.56 4930.99 8.98 4.46 46 Molineux 4 3.66 AUS4930 1.55 303.3 1s87.51 1061 .76 5.23 0.67 47 Machele 4 3.82 GS5oA 1.61 376.92 1605.57 3371 .69 4.26 2.1 48 Tahara 4 4.44 Warigal 2.15 3s.16 720.84 532.1 1 20.s 0.7 4 49 Yallaroi 4 4.74 Warigal 1.8 73.63 80.56 609.89 1 .09 7.57 50 Fallow 4 Warigal 1 .5 30 .77 93.06 375.32 3.02 4.03 51 Currency 4 4.27 AUS4930 : 1.32 98.9 38.89 277.79 0.39 7.14 52 Fallow 5 Warioal 1.48 35.16 11.11 190.13 0.32 17.11 5 3 Wariqal 5 4.52 Wariqal 1.73 165.93 31 .94 608.66 0.1 9 19.05 54 Fallow 5 Wariqal 1.67 264.83 443.06 3503.79 1.67 7.91 55 Yallaroi 5 3.86 GS50A 1.5 175.82 72.22 1425.96 0.41 19.7 4 56 Echidna 5 4.24 Wariqal 1.78 459.34 1381 .9 6 10347.2 3.01 7.4e 57 Molineux 5 3.87 Warigal i 1.54 253.85 6337.55 16810.3 24.97 2.6s 58 Grimmett 5 3.56 Warigal 2.18 85.7'l 733.34 3530.96 8.56 4.81 59 Spear 5 5.1 5 Wariqal 2.31 159.34 : 2413.91 3639.6 15.15 1.51 6 0 Canola 5 0. 43 AUS493O 98.9 592.61 61 Currency 5 3.88 GS50A 1.68 31 .87 158.33 734.59 4.97 4.64 62 Machete 5 3.71 Warigal 2.2 169.23 384.73 1316.08 2.27 3.42 63 Tahara 5 4.3S AUS4930 1 .43 39.56 126.39 734.59 3.1 9 5.81 64 Linseed 5 0.53 Warigal 2.24 70.33 187.5 759.28 2.67 4.05 65 2.77 GSSOA GS50A I 49.45 734.73 1 760.54 1 4.86 2.4 6 6 Warioal 6 4.1 4 AUS4930 1.48 'f .4 108.33 867.92 57 8.01 Appendix B 203 67 GS5oA 6 2.7 Wariqal 1 .96 152.75 288.89 3079.09 1.89 10.66 68 Tahara 6 3.76 AUS4930 1.38 558.24 234.72 522.24 0.42 2.22 69 Molineux 6 4.44 AUS4930 1.37 746.15 766.67 1392.63 .1 .03 1.82 70 Currencv 6 3.89 Wariqal 1.78 178.02 401.39 7554.52 2.25 18.82 71 Fallow 6 Warioal ' 1.48 49.45 56.94 277.79 1.15 4.88 72 Spear 6 5.05 Wariqal 1.45 32.97 30.56 101 .24 0.93 3.31 73 Linseed 6 0.79 Warioal 1.63 68.13 151.39 354.33 2.22 2.34 7 4 Grimmett 6 Wariqal 2.05 46.15 152.78 859.28 3.31 5.62 75 Yallaroi 6 3.88 Warigal 1.93 263.74 52.78 31 s0.7 0.2 s9.7 7 6 Machete 6 3.43 Wariqal 1.88 252.75 : 3350.03 6056.95 13.25 1 .81 7 7 Canola 6 Wariqal 1.32 241 .76 863.9 8308.86 3.s7 9.62 78 Echidna 6 4.14 GS50A 1.51 1012.09 2625.02 2637.11 2.59 1 79 Canola 7 0.29 Warioal 1.31 1335.16 1533.35 6876.72 1.15 4.48 80iSpear24.39 GS5OA 1 .79 1 2 1 .98 17 43.07 237 5.37 1 4 .29 1 .3 6 81 Wariqal 7 4.47 Wariqal 2.07 618.68 6600.05 10933.6 10.67 1.66 82 Yallaroi 7 4 AU5493O 1.42 84.62 436.'t 1 1906.22 s.15 4.37 83 Tahara 7 4.21 AUS4930 1.47 18.68 65.28 325.93 3.49 4.99 84 Linseed 7 o.72 AUS4930 1 .1 3 1 40.66 219 .45 544.46 1 . s6 2.48 8 5 Grimmett 7 3.53 GS50A , 1.72 136.26 108.33 2328.46 0.8 21 .49 16 Fallow 7 Warigal 1.82 30.77 120.83 337.05 3.93 2.79 87 GS50A 7 2.65 GS50A i 1.81 90.11 822.23 4000.1 9.12 4.86 8 8 r Echidna 7 4.06 AUS4930 L 1.31 200 238.89 1 1 13.61 1 .19 4.66 89 I Machete I 7 , 3.38 AUS4930 1 .1 6 429.67 37.5 2266.73 0.09 60.45 90 Currencyi 7 3.47 GS50A 1.68 s87.91 745.84 888.91 1.27 1.19 91 Molineux 7 4.26 Warioal 2.08 '129.67 72.22 2118.57 0.56 29.33 92 Machete 7 AUS4930 1.4 27.47 613.89 1 196.33 22.35 1.95 9 3 Echidna 8 4.29 AUS4930 1.51 18.68 0 139.51 0 14 Tahara I 4.29 Warigal 1.94 26.37 1 6.67 380.26 0.63 22.82 95 Fallow I Warigal 1.49 0 19.44 345.69 17.78 9 6 Canola I 0. 54 AU54930 1 .21 1 2.09 1 88.89 822.24 1 5.63 4.35 97 Grimmett: I 3.5 Warioal 1 .8 9.89 63.89 1618.56 6.46 25.33 98 Wariqal 8 3.67 Warioal 1.72 79.12 5125.04 6412.51 64.77 1.25 99 Molineux 8 2.77 Warioal 1.22 857 .14 12561 .2 1't167 14.65 0.89 1 00 Spear B 3.1 1 GS50A 1.61 1654.94 13077.9 4566.79 7 .9 0.3s 101 GS50A I 3.12 Wariqal 1.56 1873.62 879.17 7747.12 0.47 8.81 1 02 Linseed I 0. 92 AUS4930 1.37 1 169.23 2531.96 1392.63 2.17 0.55 1 03 Yallaroi 8 4.1 I AUS493O 3228.57 2972.25 2584.02 0.92 0.87 104 Currencv I 3.5 AUS4930 1.25 196.7 326.39 1930.91 1 .66 5.92 105 Molineux I 3.91 GS50A 1.71 12.09 208.34 3200.08 17.24 .t s.36 106 Grimmett I 3.6 Warioal 2.42 21.98 184.72 302.48 8.4 1.64 107 Warigal I 4.7 AUS4930 1.22 91 .21 187.5 3059.34 2.06 16.32 108 Fallow I Warioal 1.93 687.91 354.17 696.31 0.51 1.97 109 Machete I 3.67 GS50A 1.96 46.15 200 886.44 4.33 4.43 1 10 GS50A I 2.83 AUS4930 1.52 200 361.11 1659.3 1.81 4.59 111 Echidna 9 4.01 Warioal I 1.84 906.59 1375.01 7271.79 1.52 5.29 112 Yallaroi 9 3.91 AUS4930 1.62 258.24 325 1 106.2 1.26 3.4 113 Tahara I 4.35 GS50A 1.74 209.89 361.11 859.28 1.72 2.38 114 Currency I 4 Wariqal 2.02 1 3 . 1 9 65.2 8 1944.5 4.95 29 .7 9 115 Linseed 9 0.86 GS50A 1.34 568.13 21't.11 944.47 0.37 4.47 116 Canola I 0.46 GS50A 1.57 s.49 213.89 1000.03 38.93 4.68 117 Spear L 4.35 Wariqal 2.11 159.34 619.45 4363.08 3.89 7.04 118 Wariqal 10 3.51 GS50A 1.67 24.18 90.28 2400.06 3.73 26.59 119 Spoar 10 3.85 AUS4930 I 1.35 147 .25 93.06 1 361 .76 0.63 1 4.63 120 Echidna 10 4.11 Warioal , 1.75 682.42 1't29.18 22799.4 1.65 20.19 121 Grimmett 10 3.29 AU34930 0.95 2184.61 3402.81 1.56 122 Yallaroi 1O 4.16 Warioal 1.81 760.44 2058.35 14059.6 2.71 6.83 123 Cunency I 10 3.66 Warioal 2.13 246.15 604.17 12475.6 2.45 20.65 124 Machete 10 2.69 Wariqal ) 1.26 4230.77 4170.87 17301 .7 0.99 4.15 125 Fallow 10 Warioal , 1.56 0 600 3322.3'l 5.54 126 Molineux 10 4.29 Wariqal 2.31 241 .76 797.23 1303.74 3.3 1.64 127 Tahara 10 4.43 Warioal 2.21 39.56 111.11 462.98 2.81 4.17 128 Linseed 10 0.78 AUS4930 1.26 61 .54 156.95 859.28 2.55 5.48 129 Canola 10 0.18 GS50A 1.83 29.67 333.34 692.61 11.23 2.08 130 GS50A 10 2.93 Warioal 2.32 18.68 338.89 1422.26 18.14 4.2 204 Appendix C Appendix c : Preliminary rnvestigations into the Molecular Distinction of P . thornei andl P. neglectus. 205 Appendix C Appendix C Preliminary fnvestigations into the Molecular Distinction of P . thornei and, P. neglectas. C1.0 Generallntroduction The similar morphology of nematodes means that it is often difficult to identify species apart using morphological characters (Curran and Robinson, 1993). To overcome this problem, alternative diagnostic characters have been sought for identifying species and interspecific groupings within economically important nematode genera. Precise, reliable and rapid identification of economically important plant parasitic nematodes (eg Meloidogyne, Globodera, Heterodera and Ditylenchus) is an increasingly important 'Webster, component of plant protection (Curran and 1987). Over the last three decades, methods of analysing protein, lipids, carbohydrates and most recently DNA are being used as technical solutions to these taxonomic problems (Curran and Robinson, 1993). Techniques to detect DNA sequence variations between organisms can be divided into at least four basic approaches : detection of restriction fragment length polymorphisms (RFLP's) between homologous DNA sequences, use of DNA probes in dot blots and DNA sequencing (Curran and Robinson, 1993), and the polymerase chain reaction (PCR). Both root lesion nematodes, P. thornei and./or P. neglectus are found in the majority of soil samples within cropping regions of South Australia (Ch. 3.). The fact that wheat resistance differs for the two species, reinforces the importance of accurate taxonomic identification. Abad (1994) noted that in some cases the use of morphological characters is insufficient for identification because some of the definitive characteristics may overlap. As mentioned in Appendix D, the main morphological character, yVo (position of the vulva), distinguishing the two Pratylenchus species is not always 206 Appendix C definitive and can overlap between the two species. Field workers, who usually do not have the time or expertise to identify species beyond the dissecting microscope, are routinely confronted with such taxonomic difficulties. In order to address this problem, preliminary investigations were carried out to assess a molecular approach to identify P. thornei and P. neglectus. To do this, different methods of extracting DNA from nematodes were examined followed by an attempt to identify molecular differences between the DNA from P. thornei and P. neglectus . C.1.1 Extraction of DNA from nematodes C1.1.1 Introduction Investigations of different methods were made in order to obtain the best DNA extraction procedure for both species. The first extraction method is commonly used to isolate DNA from insects (O. Schmidt, pers. comm.) and did not involve purification by CsCl gradient centrifugation. In extraction method 2, two different buffers (A and B) were used followed by either CsCl gradient centrifugation or phenol/chloroform extraction. Buffer A has been used for plant DNA extractions, while buffer B is known to be an efficient fungal DNA extraction buffer (D. whisson, pers. comm.). Cl.l.z Materials and Methods P. thornei and P. neglectus were cultured on pure carrot using the modified method by Moody et al. (1973). Preparation and purification of nematode DNA The nematodes were removed from carrot cultures as previously described in Materials and Methods (Section 3.1). Both adults and juveniles were collected and rinsed at least three times in DDW. The two species were placed separately in 1.5m1microcentrifuge tubes to the lml mark which gave 300-500 thousand nematodes. The DNA was extracted from the nematodes by the following methods; 207 Appendix C Extraction method I The nematodes were pelleted (320 g,2 min.) and gxcess water removed with a pipette. The nematodes were homogenised in the tubes with 400p1 SDS buffer (10mM Tris-HCl pH8, l0mM EDTA, 17o sodium dodecyl sulfare) using a microcentrifuge pestle for 2-3 minutes at room temperature. 5¡rl of proteinase K (20mg/ml) was added and the tubes were incubated overnight at 40'C. The following day the solution was extracted with 400p1 of phenol (equilibrared with 0.lM Tris-HCl, pH8), left at room temperature for 5 minutes and centrifuged at 12 0009 for 5 minutes at room temperature to separate the two phases. The aqueous layer was transferred to a clean microcentrifuge tube and re-extracted with phenol, followed by extraction with an equal volume of chloroform. The aqueous layer (350p1) was transferred to another 1.5m1 microcentrifuge tube and the DNA was precipitated from the aqueous phase by the addition of l5pl of 5M NaCl and two volumes (730p1) of cold LO\Vo ethanol, followed by several inversions to mix and then placed on ice. After 20 minutes, the DNA was pelleted (12 0009, 10min.), washed withTTVo ethanol and resuspended in 50¡tl of rE buffer (pH 8). The DNA extracts were stored frozen at -20"C. Extraction method 2 The second extraction method involved assessing the efficiency of two different extraction procedures and purification by CsCl gradient centrifugation as compared with method 1. P. neglectus was extracted with Buffer A (2 x SSC, 20mM EDTA, 2Vo sarcoslI, 50mM Tris, pH 8), while P. thornei was extracted with Buffer B (2 x sSC, 20mM EDTA, 2vo sarkosyl, 150mM sodium acetate, pH 5.a). P' thornei and P. neglectus (f.wt. 1.3g) were frozen in liquid nitrogen and ground to a fine powder with acid-washed sand using a mortar and pestle. The powder was then brushed into lml of extraction buffer A or B containing O.2mg/ml predigested pronase and 0.05mg/ml of proteinase K and mixed gently. The contents were then placed in a l.5ml microcentrifuge tube and made to a final volume of 1.3-l.5ml with the appropriate buffer. The solution was incubated at 37"C for 30 minutes followed by 208 Appendix C 65oC for another 30 minutes. The samples were divided into two and the following extraction procedures applied. CsCl gradient centrifugation 50pl of DNA solution was placed into separate 1.5m1 microcentrifuge tubes and buffer (20mM EDTA, 50mM Tris, pH 8) was added to make a final volume of 1.4m1. CsCl (1.4g) was added and allowed to dissolve. The solution was transferred into a 3.5m1quickseal tube and 80pl of lOmg/ml ethidium bromide was added and mixed. The tubes were heat sealed and centrifuged at 272 0O0g at 20"C for 18 hours in a Beckman TL-100 ultracentrifuge. The DNA band was removed using a syringe, placed in a 1.5m1 microcentrifuge tube and made up to 250p1 with TE buffer (pH 8). The ethidium bromide was removed by the addition of 250p1 water sarurated n- butanol followed by gentle inversion. The bottom aqueous layer was pipetted into a clean 1.5m1 microcentrifuge tube and 2 volumes of TE buffer (pH S) was added followed by 2 volumes of IOOVo ethanol to precipitate the DNA. The DNA was pelleted (12 000g, 5 min.) and washed as described previously and stored frozen at -20"c. Phenol / Chloroform extractions The extracted DNA (750p1) from both species was placed into separate 1.5m1 microcentrifuge tubes and 750pL phenol was added, mixed by gentle inversion, and centrifuged at 14 0009 for 5 minutes. The upper aqueous phase was removed to a clean microcentrifuge tube and extracted with phenol as above. The aqueous phase was then extracted 2 times with 750¡rl of 24:l chloroform:isoamylalcohol to remove residual phenol. Two volumes of cold absolute ethanol were added and the samples left overnight at -20"C to precipitate the DNA. The DNA was pelleted (I2 000 g, 5 min.) and washed with lml of 70Vo ethanol containing 10mM magnesium acetate. The ethanol was poured off and the DNA pellet left to dry. The DNA was dissolved on 50pl of TE buffer (pH 8) and stored frozen at -20"c. 209 Appendix C Gel Electrophoresis of undigested DNA The amount of DNA in each sample was measured using a UV spectrophotometer. Undigested total DNA (0.2þÐ from each preparation was mixed with 2tr.l loading buffer (0.O5Vo bromophenol blue, 3Vo glycerol) and 16pl SDW to make 20p1. 10pl was removed and electrophoresed on a IVo agarose gel at 70V for 2 hours in TAE running buffer (0.04M Tris, 0.02M sodium acetate and 0.001 EDTA, pH 7.S). Hindllldigested lambda DNA was used as a size marker. The gel was stained with ethidium bromide to visualise the nucleic acids and photographed by 354nm transmitted irradiation on a transilluminator. C1.1.3 Results and Conclusions Both extraction procedures yielded DNA of high molecular weight (Fig. C.1). Extraction method 1 showed a clearer DNA extraction band. Buffers A and B used in extraction method 2 in combination with the more 'rigorous' grinding procedure gave DNA that was contaminated, possibly with protein and carbohydrates that prevented the DNA from going into solution. However, the yield of DNA was higher using the CsCl gradient which is evident from the ethidium stained gel (Fig. C.1, Lanes 8-11). The CsCl extraction used for both P. thontei and P. neglectus eliminated the RNA (lower molecular weight bands on the gel, Fig. C.1). Cl.2.l Introduction Digestion of genomic DNA with restriction enzymes generates a unique set of different sized DNA restriction fragments dependent upon the nucleotide sequence of the genome. Nucleotide substitutions, insertions or deletions that create or destroy restriction sites modify the restriction profile and therefore generate restriction fragment length polymorphism (RFLP) (Abad, 1994). The RFLPs in repetitive DNA and other high copy number sequences such as mitochondrial DNA and ribosomal DNA can be 210 Appendix C detected by agarose gel electrophoresis and visualised by ethidium bromide staining of the DNA fragments under UV light (Curran and Robinson, 1993). RFLPs have been used successfully to distinguish the two closely related potato cyst nematodes, Globodera rostochiensis and G. pallida as well as other plant parasitic nematodes including Meloidogyne and Heterodera glycines (Abad, 1994). To determine the degree of relatedness of particular fragments the homology between the bands should be established (Curran and Robinson, 1993). This can be done by hybridising labelled DNA fragments to size fractionated DNA transferred to a solid support such as a nitrocellulose filter, allowing for the visualisation of low copy number sequences (Curran and Robinson, 1993). In such cases, hybridisation of plant parasitic nematode DNA will identify the organism for which the probe is specific and as a result are quite important tools that can be used in positive or negative assay for nematode races, species or pathotypes (Abad, 1994). The initial RFLP analyses of P. thornei and P. neglectur were done using restriction enzyme digested of total DNA of P. thornei and P. neglectus probed with labelled P. thornei digested of total DNA. This was done in order to determine the degree of DNA homology between the two nematode species. The rationale for this experiment was that the repetitive DNA present in the probe would be in higher copy number and should therefore be detected on Southern blots when hybridising to DNA fragments representing repeated sequences. 211 Appendix C C.L.2.2 Materials and Methods AIu lDigest Approximately O.2¡tg of P. thornei and P. neglectu.s DNA was digested in a total volume of 60pl with 10 units of AIuI (Boehringer Mannheim @) according to the manufacturers directions. Twelve and 48pl aliquots were removed from the Alu I digests and made up to 51.5p1 with 2pl loading buffer (O.O57o Bromophenol Blue, 3OVo Glycerol) and srerile DDW. 15pl was removed from each and loaded on a l%o agarose gel containing ethidium bromide (lpgirnt). The gel was run for I hour at 70V in TBE (0.09M boric acid, 0.009M Tris base, 0.01M EDTA pH 8). 0.5pg of Boehringer Mannheim @ no 4 (lambda DNA with pSPTBM20 DNA digested with Sry I and Sau I) was used as a marker. The gel was photographed at 354nm transmitted irradiation. Multi-endonuclease Digest Five pg of both P. thornei and P. neglectus total DNA extracted from the CsCl gradient were digested in a 50pl volume with 15 units of the following Boehringer Mannheim@ restriction enzymes: PstI, Hind III, Hae II, Cla I, Bam HL Xba I and Eco RI according to the manufacturer's directions. The following day another 5 units of each enzyme was added and digestion was continued at 37"C for another hour. Restriction endonuclease digested DNA samples (50¡rl) were mixed with 10pl volume loading buffer (O.05Vo Bromophenol Blue, 30Vo Glycerol) and 15pL was placed in slots of a lvo agarose gel containing ethidium bromide (lpg/ml) along with 0.5pg of Boehringer Mannheim @ no 4 marker. The gel was electrophoresed at 50V overnight in TAE running buffer (pH 7.8) and photographed at 354nm transmitted irradiation. Southern Hydridization Both the AIu I digest and the multi-endonuclease digests (CL.2.2) were transferred from the IVo agarose gel to nylon filters by Southern blotting 212 Appendix C as described by Sambrook ¿t aL (1989). The filter containing the multi-endonucleased digests of P. thornei and P. neglectu,r was hybridised with ¡32P1-dCTP labelled Alul digested P. thornei DNA as the probe. The labelled probe was generated using the Megaprime DNA labelling system RPN 1606 (Amersham@) according to the manufacturer's protocol. The filter was prehybridized for 2 hours at 65'C followed by hybridisation at 65oC overnight (Sambrook et a\.,1989) in a rolling bottle hybridisation oven (Hybaid). The unbound probe was poured off and the filter washed twice in 2x SSC + 0.17o SDS for 20 minutes at 65'C. This was followed by two more 20 minutes washes in 0.2x SSC + 0.17o SDS also at 65oC. The filter was then exposed to X-ray film for 16 hours at70"C and developed. C1.2.3 Results RFLP Some band differences were evident for P. neglectøs and P. thornei for some of the restriction endonucleases investigated (Fig. C.2), in particular XbaI and Eco RI (Lanes 16-19). This suggests that highly repetitive DNA sequences differ between the two species. However, the bands wero not clearly distinguishable between species within the range of endonucleases examined. To further verify the possible banding distinctions between species Southern blot hybridisations were performed using the radioactive labelled AIuI digested P. thornei DNA as a probe. This digest was used as a probe because small DNA fragments are necessary for efficient labelling. H)¡bridisation The radiolabelled P. thontei DNA probe hybridized to both P. thornei and P. neglectus digests. Some restriction enzymes, in particularXba I (Lanes 16 and 17, Fig. C.3.) identified repetitive DNA bands in P. thorn¿i, which were absent in P. neglectus However the DNA was not sufficently digested (Fig. C.2), hence the experiment would need to be reconfirmed with completely digested DNA. Aooendix C Fig C.1 Agarose gel separation of P. thornei and P. neglectus total undigested DNA. Ethidium bromide stained gel viewed under 354nm transmitted UV inadiation: Lane 2, Extraction I P. thornei; Lane 3, Extraction I P. neglectus, Lane 4, P. neglectus Extraction 2 (buffer A, phenol); Lane 5, P. thornei Extraction 2 (buffer B, phenol); Lane 6 and 7 repeat of Lane 4 and 5; Lane 8, P. neglectus Extraction 2 (buffer A, CsCl); Lane 9, P. thornei Extraction 2 (buffer B, CsCl); Lane 10 and 11 repeat of Lanes 8 and 9; Lanes 1 and 12, Hind III digested lambda DNA (in single basepairs indicated at right). fig C.2 Agarose gel separation of P. thornei and P. neglectus of DNA digested with restriction endonucleases. Ethidium bromide stained gel viewed under 354nm transmitted UV irradiation showing the fragment size distribution of the 7 restriction endonucleases: Lane 2, Pst I P. neglectus;Lane 3, Pst I P. thornei;Lane 4, Hindlll P. neglectus; Lane 5, Hind III P. thornei; Lane 6, Hae Il P. neglectus; Lane 7 , Hae lI P. thornei; Lane 8, CIaIP. neglectus; Lane 9, CIaIP. thornei; Lane lI Bam HI, P. neglectus;Lane 12, BamHI P. thornei; Lane 13, ClaI P. neglectus;Lane 14, CIa I P. thonteii Lane 15, Cla I P. thornei; Lane 16, XbaI P.neglectus; Lane I7, Xba I P. thorneia Lane 18, Eco RI P. neglectus;Lane 19, Eco RI P. thornei. Lane 1 and 10, DNA marker, Boehringer Mannhein @ no. 4 (size in basepairs indicated on right). I 2 i ,+ 5 (¡ 7 8 9 l0 Ii 12 + 23t30 941 6 655'7 4361 ¿Ë{ a1a1 2021 I ËËf 564 t I ) .3 + -5 (r 7 f3 9 l0 ll tl l] I+ I.t 16 11 lll 19 1932!l 11 4'J 5 526 4254 3 140 2690 2322 t822 1489 1150 Anoendix C Fig. C.3 : Autoradiograph of Southern blot showing hybridization of P. thornei [32p]- dCTP labelled AIu I digested P. thornei DNA to the multi-endonuclease digest of both nematode species (see Fig. C.2). | 2 3 '1 -j 6 7 8 9 t0 ll 12 13 t4 t.s t6 t] t8 t9 2r3 Appendix C C.1.2.4 General Conclusion Although RFLP data is valuable in preliminary studies, usually relatively large quantities of DNA are required (0.l-lpg per sample) and the time consuming gel electrophoresis, transfer and hybridisation steps preclude this approach when processing large numbers of samples in a routine diagnostic assessments (Curran and Robinson, 1993). Preliminary evidence is presented here to indicate that DNA polymorphisms are evident between P. thornei andP. neglectus, although the band differences were not resolved sufficiently due to the poor quality of the CsCl prepared DNA. However, hybridisation differences between nematode species have been detected, suggest the possibility of using species-specific probes. To obtain specific probes several possibilities exist. A phage library may be constructed with genomic DNA of P. thorn¿i. This could then be hybridised against the Alu digest of both species and screened for clones with strong signals. These clones would then need to be verified as species-specific by hybridisation studies using genomic DNA of both species. If species specificity exists it may be possible to sequence them and develop PCR primers. An alternative method is to use a genomic library to subclone some of the gel eluted fragments. The fragments would need to be cut out of the gel, ligated into an appropriate vector and hybridised with genomic DNA from both species to verify species-specific fragments. It is once again possible to sequence the DNA and develop PCR primers. Such a PCR based technique has been developed to identify individual nematodes in the genus Pratylenchøs (Samas and Linden,1994). This technique is simple and rapid, producing differential reproducible banding patterns between P. penetrans, P. scribneri, P. hexincisus and P. agilis. Curran et al. (1993) are developing species specific probes for many species of Pratylenchus with the possibility of extracting 'PCR' grade DNA 214 Aooendix C from soil and plant tissues in addition to the quantitative assessment of these nematodes. The success of the development of such molecular techniques for diagnostic identification, detection (pre-planting) and quantification of nematode populations will be influenced by the economic viability of the assay. This will be determined by commercial considerations such as market size and regulatory requirements (Curran and Robinson 1993). However, it is likely that the importance of biotechnology-based methods for diagnosis will increase as management systems are developed to fully utilise the management practices, such as the use of resistant and tolerant cultivars in nematode problems (Curran and Robinson, 1993). 215 Appendix D Appendix D: Morphometrics of south Australian population of P. thornei andP. neglectus males and females Please note the first two authors a¡e the primary contributors. This Appendix is currently In Press. 216 Appendix D Description of the Male and Redescription of the Female Pratyle nc hu s thorn ei and P røtyle nc hus ne gle ctus from Australia. Abdolhossein Taheril, Julie M. Nicol2, Janine Lloyd2 and Kerrie A. Davies2 lDepartment of Plant Science, University of Adelaide, Waite Campus, PMB 1, Glen Osmond, South Australia, 5064 'Waite 2Department of Crop Protection, University of Adelaide, Campus, PMB 1, Glen Osmond, South Australia, 5064. D1.0 Abstract The first descriptions of male and female Pratylenchus thornei and Pratylenchus neglectus from Australia are presented, based on scanning electron microscopy (SEM) and light microscopy. Nematodes from populations of P. thornei and P. neglectus found in South Australia are similar to those previously described from Europe, Africa, North America and the United Kingdom. As reported by other workers, there is considerable variation and overlap of measurements, making it difficult to determine suitable taxonomic characters to distinguish the two species. Body length, wlval percentage and number of lip annules are considered the most important characters by which to distinguish the two species. D 1. 1 Introduction Members of the genus Pratylenchus (Nematoda: Pratylenchidae) parasitise a wide variety of plants (Loof, 1991). Pratylenchus thornei (Sher and Allen, 1953) and the 217 Appendix D related species P. neglectus (Rensch,1924) have a cosmopolitan distribution (Loof, 1991) and are also widespread in South Australia (Nicol, unpublished data), often having overlapping distributions. They cause yield losses of wheat in glasshouse tests and in the field (Thompson et al., 1981; Doyle et al., 1987; Nicol, I99l: Taheri et al., 1994). The difficulty of identifying Pratylenchøs species in Australia is a major impediment to sound ecological studies (Stirling and Stanton,1993). P. thornei and P. neglectus are parthenogenetic and males although found are rare. Several were collected from cultures which allowed a more detailed description of males than has previously been published. Descriptions and morphometric measurements of P. thornei and P. neglectus from Australia were made to compare Australian populations with those from other countries, and to determine which are the most useful characters for Australian field workers to use to distinguish the two species. Dl.2 Materials and Methods Laboratory cultures of P. thornei and P. neglectus were reared on aseptic carrots by a method modified from Moody et al.(1973). Field populations of both species were derived from cereal and legume fields in South Australia, and extracted from soil using a modified Baermann technique. Nematodes were killed and fixed in hot (100"C) formalin/acetic acid 4:1. Specimens for light microscopy were processed by slow evaporation through ethanol - glycerol at 40'C over 2 weeks, mounted in glycerol on permanent slides, and examined using a Nomarski microscope. Nematodes for SEM were dehydrated in an alcohol series, critical point dried, coated with 30nm of gold and examined under 20kV using a Cambridge 5250 microscope. All measurements have been rounded to the nearest whole number. 2r8 Appendix D D 1.3 Descriptions Pratylenchus thornei (Figs. 1,3 & 4) Measurements: Table 1. Females. As per decription by Fortuner (1977). Males. Body forming a very open "C" shape when killed. Cuticle with fine inconspicuous transverse striae, appears smooth in some specimens in light microscope. Body annules 1.9pm wide (1.5-2.3¡tm). Lateral tield with four incisures. Three lip annules, continuous with body outline. SEM of the lip region showed oral disc fused to sub-median segments which broadened towards outer edge. Amphid openings rounded, on the inner edges of the lateral segments. Outer margin of sclerotized labial framework extends about two annules into body and one annule into lip region. Stylet guiding apparatus extends posteriorly from basal plate for about four annules. Stylet medium size (13-19pm long) with broadly rounded to almost anteriorly flattened basal knobs. Orifice of the dorsal oesophageal gland about 4pm behind stylet base. Nerve ring directly behind oesophageal bulb. Excretory pore opening 59 to 84pm behind head. Hemizonid about two annules long, one annule anterior to excretory pore. Oesophageal glands in one lobe,29 to 47¡tm long, extending longitudinally and ventrolaterally over intestine. Outstretched testis with spermatocytes in a single row, followed by a region of multiple rows. Phasmids slightly posterior to mid-tail, not extending to edge of bursa. Bursa sometimes shorter in region of phasmid, edges smooth; peloderan. Spicules l8 to 2l¡tm; arcuate, hafted. Gubernaculum trough-shaped. Tail dorsally convex-conoid, terminus bluntly rounded to truncate, unstriated. Collection sites. Field specimens Triticum aestivum, Tarlee, South Australia. Nematodes in carrot cultures were originally collected from Waite Agricultural Research Institute soils, Urrbrae, South Australia. Voucher specimens. Specimens deposited in the Waite Institute Nematode Collection (WINC), Adelaide, South Australia. Field specimens are numbered 6614 and 6618, and specimens from cultures are 8168. MorPhonl¿trtcs oI the male and female P. Ihornc¡ and P. ncglectus collected Írom laboratory and field populatíons ín South Att¡tralia. Fanqþs Malcs P. thorrci P. ruglcctus P. thornci P. ncgleaus Meauanør(¡rn) Field Culh¡re Field C¡l¡üc Field Cuttr¡¡c Field G¡l¡¡¡e n n n n n n n n ' Body l,ørgth 7 522 2t 691 l4 483 20 475 3 515 9 566 I 431 3 431 471æ 6tù774 42t-524 425-503 4E8-557 4ll{18 42ß-432 An¡cria o 7 15 E6 l4 E E¡crclcry Porc 7E 90 l8 75 3 73 3 6E 74-84 8G91 82-95 7t-79 59-E4 670 Styld tÆntth 7 t1 lE t7 l4 18 20 t7 3 t4 7 l7 l6 3 16 lGlS 1618 17- 19 1619 l3-15 lG19 15-¡E \ilidrh of Sryler 7 5 Knobs 418 l4 520 5 3 4 9 4 5 3 3 3-5 4-5 5-6 4-5 44 3-5 3-4 Greá¡rr Body vÍidh 7 lE 2l 24 14 19 20 20 J l6 8 1E 20 3 1ó 1619 lE-28 r7-21 1ó24 t6tE IGI9 Widh of }ledian 7 Bulb 921 l0 l4 l0 20 l0 3 9 E 9 9 J t 7-9 9-12 9-t2 8-l I 8-l I 8-10 7-9 Tot¡l Gor¡ad not measured notmeåswed t 2n 7 317 3 2æ I.engtlt 203-255 2&-364 203-2r3 WiùhatCbacs N/A ¡{/A 3 l4 E l5 Lr23 13 t2-15 l3-17 t2-t! WiôhatVulva 7 1t 2t 24 l4 19 t7 l9 N/A N/A r5-2t 2t-26 tó2t t7-23 Lips to Vulva 7 40t 2t 514 t3 402 t7 3s9 N/A N/A 36G500 403-5E6 361-446 346411 a 7 29 2l 29 14 26 20 24 3 31 9 32 23 n n-33 ?5-t8 24.28 2G28 3G3l )'t-aa t _r, c 7 t9 17 2l l4 23 t'l a1 3 3t t 20 ló 3 22 9-29 1t-?5 2ù25 20-26 3541 1G24 2t-u c' 7 29 2l 29 l4 26 t7 u 3 3E 8 39 38 3 34 ?ß-34 ?5-33 24-2t 2t-2E 35-4t 28-47 34-15 v% 7 7E 2l 74 l3 E2 t7 82 N/A t[/A 1GE2 6't9 8GE6 75-84 T% N/A N/A t 44 E 57 J 4t 4G5l 45-t9 47-49 Lip Armrles 7 3 2t 3 l4 t 202 J 3 9 3 2 3 2 ., Height Lips 7 4 2t 3 l4 3 20 3 3 2 9 2 3 J 3-5 2-4 2-4 24 2-3 )_a 3-3 LengthPoa Vulva 6 t9 t7 25 l4 16 fi l7 N/A I'VA Sac t5-22 t4-43 tt-23 9-24 Spicule Lørgth N/A N/A 9 20 l5 t 17 lE-23 t6rt Tail L€agth 7 27 2t 33 l4 2t t7 2t 3 27 9 2t IE 3 l9 22-39 25-39 tt-23 t8-23 24-30 t-2t t¿aDLÞ If Publish¿d ûtoryhoûl¿trics of ntale andferøle P. tltornei and P. neglectus worldwíde. Females Males P. negleclus P.tlørn¿i P. P. thornci ^egleclvt Fr€derick & Handoo & D'Errico l¡of Corbett Nicol Frcde¡ick & Handæ & She¡ & Allen CoòeÍ Sher & Allen For¡¡ner lrof Sher& Allen Lrof Authon T"rjarr Golden (1970) 0e60) 0e70) (leel) Tada¡ Golden (19s3) it970) (1953) (t977) (1960) (1953) (1960) (19E9) (1989) (1989) (1989) Measuranent (¡m) Body lsrc¡h 5¿m 709 490 461 480 551 492 3& 472 4ó0610 450-n0 4544t4 40E-708 420-@0 620-825 410-530 3 l2-58E 310-550 370-450 - 420-524 Stylet længth 17 t5 t4 l7 l6 l6 16 t4 16 l5-lE l5-r9 15-19 t4-t7 l2-r8 l6-18 15-19 16-18 t6t7 - 15-17 Greates Body 25 not measu¡ed not measurcd not meesured wïdh 2ù33 Wid¡hof Mdian 23 not measured not measurcd not me¿sured Bulb 8-30 a 33 29 n 32 39 29 22 n 2634 26-36 28-32 25-36 n-37 25-3r 23-3t t6-32 r8-25 24-30 25-29 b 6 6 6 5 6 6-8 5-8 5-8 5-E 5-8 44 5-6 c 20 l3 2t 20 20 19 20 20 l9 l8-24 19-25 r9-2E t7-25 t8-n l0-17 t'l-23 t4-27 16-lE t7-21 r7-22 c not measured not meazured not meåsufed r.5-2.5 Tlo N/A N/A 30 49 42-56 Ylo 76 75 82 82 N/A N/A 75:19 74-79 76-79 74-79 76-79 80-84 75-87 80-88 7E-83 l¿æral Incisurcs 4 4 not me¿sured not me¿surcd Spicule Lengtlr N/A N/A 2l not measured ,, Lip Annules 3 3 7 not measured not mean¡red Tail Annr¡les not measured not me¿su¡ed 2ù29 t6-2t A B c D E F' 20¡tm D- 40pm Fig.l.Pratylencltusthornei"A'oesophagealregionfemale;B'tailregionfemale;C' entiremale;D,headendfenrale;E,oesophagealregionmale;F'tailregionmale' A B C EF E Ooo ôo o F 20pm CD 100¡rm AB- 20¡tm female; A, oesophageal region female; B, tail region Fig. 2. P ratylencltus negLectt'ts' C,oesophagealregionmale;D'heaclendfemale;E'tailregionnrale;F'entiremale A ts a o \ , ,¡ i ''u ,5 um lr' ç, U D - r¡J' I r 5um A' heacl prttt,t,le,clt.t,t.¡ tltr¡,l¿l and Pr,.t\¡LcnchrL,s rtcgler:tt'¿'ç [rig. 3" Light .ricroscol]y o1, thorttei C' tlrrl P neglat:ttL't rlrale; D' tall P' [) ncglcr:tr.tî nltle. B, heltrl [' lltt¡tttailèllale. nrlLlc attdPral'Lenclttts negLectu'l' A' head Fig.4. SEM nlicrographs or Prar)'Lettcht'ls tlrcrtrci tail P' neglecttt's male; D' tail P' tlrcrnei P. neglectus rrrale; B, heacl P. th.ornei.tllale; c, lateral field P' thornei fernale; G' vulva P nrale; E, lateral field P. neglectus uale; F, negLecttrs fen-rale; H, vulva P ' thornei fenlale' 2t9 Aooendix D Pratylenchus neglectus (Figs. 2,3 &, 4) Measurements: Table 1. Females. As per decription by Townshend and Anderson (1976) Males. Body assuming a straight to very open "C" when killed. Cuticle has fine, inconspicuous transverse striae. Body annules 1.7pm wide (1.3-2.3). Lateral field with four lines, areolation of all bands seen with SEM. Head with two annules about equal size, the apical one comprising the lips. SEM showed oral disc fused to sub- median segments which broadened at outer edges, forming a distinct "head cap". Amphid openings small and sliçlike, seen with SEM on inner edges of lateral segments. Stylet medium size (15-18pm long). Stylet knobs 3 to 5pm across, typically indented on anterior surfaces. Dorsal gland orifice 3 to 5pm posterior to stylet. Nerve ring directly behind oesophageal bulb. Oesophageal glands in one small lobe, 15 to 17pm long, extending longitudinally and ventrolaterally over intestine. Excretory pore 66 to 70pm from head end. Hemizonid immediately anterior to excretory pore, extending two or three body annules. Outstretched testis with spermatocytes in single row, followed by a region of multiple rows. Phasmids slightly posterior to mid-tail, extending to near the edge of bursa. Edges of bursa are smooth at tail tip but crenulated near the point of origin; peloderan. Spicules 15 to 18pm long, hafted; gubernaculum arcuate. Tail terminus without annulation, bluntly rounded to truncate. Collection sites. Field specimens from Triticum aestivum, Minippa, South Australia. Nematodes in carrot cultures were obtained from Dr. V. A. Vanstone, University of Adelaide, who originally collected them from field soil, Palmer, South Australia. Voucher specimens. Specimens deposited in the WINC, Adelaide, South Australia. D 1.4 l)iscussion Males. In more than half the described species of Pratylenchus males are infrequent, rare or unknown (Sher and Allen, 1953). Until recently, only three specimens of male P. neglectus had been described (Sher and Allen, 1953; Loof, 1960) and similarly for p. 220 Appendix D thornei (Sher and Allen, 1953; Fortuner,1977; Loof, 1960). Vovlas and Castillo (1995) reported finding 2 males for every 1000 females of P. thornei grown on carrot disc cultures. Similar ratios have been observed for both P. thornei and P. neglectus grown on carrot cultures in our work. Morphometrics of the Australian isolates are comparable to those previously documented (Table 2). The basal knobs of the stylet of Australian P. thorn¿i males are broadly rounded (Fig. 1), but in P. neglectus they are typically indented on the anterior surfaces and less robust (Fig. 2). The spicule length is longer in Australian specimens of P. thornei than P. neglectus (Table 1, Fig. 3), as previously documented by Loof (1960) and Sher and Allen (1953). The caudal alae could be used to distinguish the two species (Fig. 4). However, while the bursa of. P. neglectus has crenulated edges near its origin anterior to the cloaca, and that of P. thornei is smooth, it is difficult to see this with light microscopy. The bursa tended to roll inwards during preparation for S.E.M. and this obscured the edges. The position of the opening of the phasmids on the bursa was variable, although they opened closer to the edge of the bursa in P. neglectus, and in some specimens of P. thornei the bursa appeared shorter in the vicinity of the phasmids. Females . The morphology and morphometrics of both P. neglectus and P. thornei (Table 1, Figs 1, 2) from Australia showed that the females are similar to isolates of each species from other countries (Table 2), except for the ratio c in P. neglecløs females. This was larger for the Australian isolates than for isolates from the U.S.A. (Handoo and Golden, 1989) or for the averages of published data calculated by Frederick and Tarjan (1989), suggesting that the Australian isolates had shorter tails. However, Loof (1991) stated that the number of tail annules showed a wide range within species of Pratylenchus. Length of the post-uterine sac of European specimens of female P. thornei was more than one and half times the body width at the vulva (Fortuner, lg77), but in P. neglectus the sac was less than or equal to the body width (Townshend and Anderson, 1976). The 22r Appendix D measurements reported here (Table 1) show that Australian isolates of both species have similar or equal measurements for the length of the post uterine sac and body width at the vulva. Roman and Hirschmann (1969) found that the length of the post-uterine sac was very variable within species. Characters for diagnosis of species by field workers. Body length and distance from lips to vulva of female P. thornei are significantly greater for specimens from cultures than from the field, and males from cultures were also larger than specimens from the field. Male and female P. neglectus from carrot cultures and the field are similar in size (Table 1). De Man's ratios confrrmed that there was little variation in the morphometrics of these two species whether from cultures or the field (Table I ). Roman and Hirschmann (1969) found extensive morphological variation in P. vulnus from greenhouse cultures compared with specimens from callus cultures. This may reflect host physiology or nutritional status of the nematodes. Loof (1991) stated that, in general, Pratylench¿rs spp. extracted from roots are longer and stouter than specimens extracted from soil, and Olowe and Corbett (1984) found that body length was greater on favourable than on unfavourable hosts. The size difference of P. thonnei fromthe field versus carot cultures, which was not seen for P. neglectus, may suggest that carrots are a more suitable host for thornei than for neglectus. In general, Australian workers could assume that adults of P. neglectus from the field would be smaller than those of p. thornei, but body size alone would not be diagnostic. Corbett and Clark (1983) suggested that the number of lip annules was a reliable character distinguishing the two species. This study confirmed that P. neglectu.r has two offset head annules (Handoo and Golden, 1989; Corbett, I}TO), while P. thorneihas three (Sher and Allen, 1953; Corbett, 1970) which are continuous with the body (Figs. 3, 4, Table 2). However, this character can only be checked using an oil immersion lens. 222 Appendix D Australian specimens of both species have four incisures in the lateral field of both sexes (Fig. 4), as reported by Sher and Allen (1953) and Handoo and Golden (1989). In Pratylenchur spp. the lateral field starts on the seventh to ninth body annule and for the greater part of its length has four lines or three bands (Corbett and Clark, 1983). However, across or within the bands there may be further lines which are said to be characteristic for each species but are found to vary greatly, as did the distance and depth of the transverse striae. This was particularly so for P. neglectu.r where complete or partial areolation of the middle band has been found. In the Australian specimens, male P. neglectus showed areolation of all three bands in SEM, but the bands of P. thornei (in both species) were smooth and no transverse striae were seen. However, these differences in the lateral fields cannot be seen with the light microscope, and thus, the lateral fields cannot be used by field workers to separate the two species. Males of both species had body annules with similar widths. Corbett and Clark (1933) found rhat p. thornei females, with average annule width of 1.4pm, were more finely annulated than P. neglectus, with 1.6pm, and that the transverse striae of the latter were deeper than in P' thornei which sometimes looked as if the cuticle was smooth. This was also true of the striae of the Australian isolates of the two species. Loof (1991) stated that the diagnostic value of the shape of the stylet knobs, stylet length, length of the oesophageal gland, and length of the post-uterine sac was limited due to difficulty of measurement or intraspecific variability. Number of tail annules is variable even within one species and cannot be used as a diagnostic character (Roman and Hirschman, 1969; Loof, 1991), but shape of the terminus is more reliable (Loof, I99I). The Australian specimens of thornei had broadly rounded to truncate tail ends, but those of neglectur were rounded to oblique, as described for specimens from other countnes. 223 Appendix D Current identification of both species of. Pratylenchus using light microscopy is difficult for workers in applied field research who rely heavily on body length and vulval percentage, characters which can be checked using a dissecting microscope. Study of nine morphometric characters of six Pratylenchøs species revealed vulval percentage to have the lowest coeffeciency of variation (Roman and Hirschmann, 1969). However, care should be taken in using this character to distinguish P. neglectus and P. thornei from Australia as an overlap (80-86Vo and 76-82Vo respectively) was observed, particularly with field populations. Hence, an increase in sample size may be necessary to distinguish the two species. Loof (1991) recommended that 25 specimens be measured for diagnosis. Use of the compound microscope can delineate species more accurately on the basis of the number of lip annules and head shape, but is time consuming and requires considerable technical expertise, and therefore may not be practical for field workers. There is an urgent need for development of a molecular technique to confirm visual identifications of Pratylenchus species. 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